Genetically-modified t cells comprising a modified intron in the t cell receptor alpha gene

ABSTRACT

The present invention provides a genetically-modified T cell comprising in its genome a modified human T cell receptor alpha gene. The modified T cell receptor alpha gene comprises an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene that is positioned 5′ upstream of TRAC exon 1. The exogenous sequence of interest can comprise an exogenous splice acceptor site and/or a poly A signal, which disrupts expression of the T cell receptor alpha subunit. The sequence of interest can also include a coding sequence for a polypeptide, such as a chimeric antigen receptor. Additionally, the endogenous splice donor site and the endogenous splice acceptor site flanking the intron are unmodified and/or remain functional. The invention further provides compositions and methods for producing the genetically-modified cell, and populations of the cell, and methods for the treatment of a disease, such as cancer, using such cells.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/627,052, filed Dec. 27, 2019, which is a national stage filing under35 U.S.C. § 371 of International Application No. PCT/US2018/039740,filed Jun. 27, 2018, which claims the benefit of U.S. ProvisionalApplication No. 62/527,845, filed Jun. 30, 2017 and Application No.62/579,473, filed Oct. 31, 2017, each of which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to the fields of oncology, cancer immunotherapy,molecular biology and recombinant nucleic acid technology. Inparticular, the invention relates to genetically-modified T cellscomprising a modified intron in the T cell receptor alpha gene that is5′ upstream of TRAC exon 1, as well as compositions and methods formaking the same. The invention further relates to methods of using suchcells for treating a disease, including cancer, in a subject.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 25, 2021, isnamed P109070024US03-SEQ-EPG, and is 121 kilobytes in size.

BACKGROUND OF THE INVENTION

T cell adoptive immunotherapy is a promising approach for cancertreatment. This strategy utilizes isolated human T cells that have beengenetically-modified to enhance their specificity for a specific tumorassociated antigen. Genetic modification may involve the expression of achimeric antigen receptor or an exogenous T cell receptor to graftantigen specificity onto the T cell. By contrast to exogenous T cellreceptors, chimeric antigen receptors derive their specificity from thevariable domains of a monoclonal antibody. Thus, T cells expressingchimeric antigen receptors (CAR T cells) induce tumor immunoreactivityin a major histocompatibility complex non-restricted manner. T celladoptive immunotherapy has been utilized as a clinical therapy for anumber of cancers, including B cell malignancies (e.g., acutelymphoblastic leukemia (ALL), B cell non-Hodgkin lymphoma (NHL), andchronic lymphocytic leukemia), multiple myeloma, neuroblastoma,glioblastoma, advanced gliomas, ovarian cancer, mesothelioma, melanoma,and pancreatic cancer.

Despite its potential usefulness as a cancer treatment, adoptiveimmunotherapy with CAR T cells has been limited, in part, by expressionof the endogenous T cell receptor on the cell surface. CAR T cellsexpressing an endogenous T cell receptor may recognize major and minorhistocompatibility antigens following administration to an allogeneicpatient, which can lead to the development of graft-versus-host-disease(GVHD). As a result, clinical trials have largely focused on the use ofautologous CAR T cells, wherein a patient's T cells are isolated,genetically-modified to incorporate a chimeric antigen receptor, andthen re-infused into the same patient. An autologous approach providesimmune tolerance to the administered CAR T cells; however, this approachis constrained by both the time and expense necessary to producepatient-specific CAR T cells after a patient's cancer has beendiagnosed.

Thus, it would be advantageous to develop “off the shelf” CAR T cells,prepared using T cells from a third party, healthy donor, that havereduced expression of the endogenous T cell receptor and do not initiateGVHD upon administration. Such products could be generated and validatedin advance of diagnosis, and could be made available to patients as soonas necessary. Therefore, a need exists for the development of allogeneicCAR T cells that lack an endogenous T cell receptor in order to preventthe occurrence of GVHD.

Genetic modification of genomic DNA can be performed usingsite-specific, rare-cutting endonucleases that are engineered torecognize DNA sequences in the locus of interest. Methods for producingengineered, site-specific endonucleases are known in the art. Forexample, zinc-finger nucleases (ZFNs) can be engineered to recognize andcut pre-determined sites in a genome. ZFNs are chimeric proteinscomprising a zinc finger DNA-binding domain fused to the nuclease domainof the FokI restriction enzyme. The zinc finger domain can be redesignedthrough rational or experimental means to produce a protein that bindsto a pre-determined DNA sequence ˜18 basepairs in length. By fusing thisengineered protein domain to the FokI nuclease, it is possible to targetDNA breaks with genome-level specificity. ZFNs have been usedextensively to target gene addition, removal, and substitution in a widerange of eukaryotic organisms (reviewed in Durai et al. (2005), NucleicAcids Res 33, 5978). Likewise, TAL-effector nucleases (TALENs) can begenerated to cleave specific sites in genomic DNA. Like a ZFN, a TALENcomprises an engineered, site-specific DNA-binding domain fused to theFokI nuclease domain (reviewed in Mak et al. (2013), Curr Opin StructBiol. 23:93-9). In this case, however, the DNA binding domain comprisesa tandem array of TAL-effector domains, each of which specificallyrecognizes a single DNA basepair. A limitation that ZFNs and TALENs havefor the practice of the current invention is that they areheterodimeric, so that the production of a single functional nuclease ina cell requires co-expression of two protein monomers.

Compact TALENs have an alternative endonuclease architecture that avoidsthe need for dimerization (Beurdeley et al. (2013), Nat Commun. 4:1762).A Compact TALEN comprises an engineered, site-specific TAL-effectorDNA-binding domain fused to the nuclease domain from the I-TevI homingendonuclease. Unlike FokI, I-TevI does not need to dimerize to produce adouble-strand DNA break so a Compact TALEN is functional as a monomer.

Engineered endonucleases based on the CRISPR system are also known inthe art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013),Nat Methods 10:957-63). A CRISPR endonuclease comprises two components:(1) a Cas effector nuclease, typically microbial Cas9, Cpf1, or anothersuitable nuclease; and (2) a short “guide RNA” comprising a ˜20nucleotide targeting sequence that directs the nuclease to a location ofinterest in the genome. By expressing multiple guide RNAs in the samecell, each having a different targeting sequence, it is possible totarget DNA breaks simultaneously to multiple sites in the genome. Thus,CRISPR nucleases are suitable for the present invention. The primarydrawback of the CRISPR system is its reported high frequency ofoff-target DNA breaks, which could limit the utility of the system fortreating human patients (Fu et al. (2013), Nat Biotechnol. 31:822-6).

Homing endonucleases are a group of naturally-occurring nucleases thatrecognize 15-40 base-pair cleavage sites commonly found in the genomesof plants and fungi. They are frequently associated with parasitic DNAelements, such as group 1 self-splicing introns and inteins. Theynaturally promote homologous recombination or gene insertion at specificlocations in the host genome by producing a double-stranded break in thechromosome, which recruits the cellular DNA-repair machinery (Stoddard(2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases are commonlygrouped into four families: the LAGLIDADG (SEQ ID NO: 2) family, theGIY-YIG family, the His-Cys box family and the HNH family. Thesefamilies are characterized by structural motifs, which affect catalyticactivity and recognition sequence. For instance, members of theLAGLIDADG (SEQ ID NO: 2) family are characterized by having either oneor two copies of the conserved LAGLIDADG (SEQ ID NO: 2) motif (seeChevalier et al. (2001), Nucleic Acids Res. 29(18): 3757-3774). TheLAGLIDADG (SEQ ID NO: 2) homing endonucleases with a single copy of theLAGLIDADG (SEQ ID NO: 2) motif form homodimers, whereas members with twocopies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers.

I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 2) familyof homing endonucleases that recognizes and cuts a 22 basepairrecognition sequence in the chloroplast chromosome of the algaeChlamydomonas reinhardtii. Genetic selection techniques have been usedto modify the wild-type I-CreI cleavage site preference (Sussman et al.(2004), J. Mol. Biol. 342: 31-41; Chames et al. (2005), Nucleic AcidsRes. 33: e178; Seligman et al. (2002), Nucleic Acids Res. 30: 3870-9,Arnould et al. (2006), J. Mol. Biol. 355: 443-58). More recently, amethod of rationally-designing mono-LAGLIDADG (SEQ ID NO:2) homingendonucleases was described that is capable of comprehensivelyredesigning I-CreI and other homing endonucleases to targetwidely-divergent DNA sites, including sites in mammalian, yeast, plant,bacterial, and viral genomes (WO 2007/047859).

As first described in WO 2009/059195, I-CreI and its engineeredderivatives are normally dimeric but can be fused into a singlepolypeptide using a short peptide linker that joins the C-terminus of afirst subunit to the N-terminus of a second subunit (Li et al. (2009),Nucleic Acids Res. 37:1650-62; Grizot et al. (2009), Nucleic Acids Res.37:5405-19). Thus, a functional “single-chain” meganuclease can beexpressed from a single transcript.

The use of engineered meganucleases for cleaving DNA targets in thehuman T cell receptor alpha gene has been previously disclosed. Forexample, in International Publication Nos. WO 2017/062439 and WO2017/062451, Applicants disclosed engineered meganucleases havingspecificity for recognition sequences in the T cell receptor alphaconstant region (TRAC) gene exon 1. The '439 and '451 publications alsodisclosed methods for targeted insertion of a CAR coding sequence intothe meganuclease cleavage sites. Further, International Publication No.WO 2014/191527 disclosed variants of the I-OnuI meganuclease that werealso engineered to target a recognition sequence (SEQ ID NO: 3 of the'527 publication) within TRAC exon 1. Although the '527 publicationdiscusses that a chimeric antigen receptor can be expressed in TCRknockout cells, the authors did not disclose the insertion of the CARcoding sequence into the meganuclease cleavage site.

The use of other nucleases and mechanisms for disrupting expression ofthe endogenous TCR have also been disclosed, including small-hairpinRNAs, zinc finger nucleases (ZFNs), transcription activator-likeeffector nucleases (TALENs), megaTALs, and CRISPR systems (e.g., Osbornet al. (2016), Molecular Therapy 24(3): 570-581; U.S. Pat. No.8,956,828; U.S. Publication No. US2014/0301990; U.S. Publication No.US2012/0321667).

Additionally, Eyquem et al. ((2017), Nature 543: 113-117) disclosed theuse of a CRISPR/Cas9 system to target insertion of a CAR coding sequenceinto a site that spans both the 5′ end of TRAC exon 1 and the endogenoussplice acceptor site that is positioned immediately 5′ upstream of TRACexon 1. The authors describe that the expected double-strand cleavagesite of the Cas9 nuclease is within the splice acceptor site (see,Eyquem, Supplementary FIG. 1A). This splice acceptor site is necessaryfor TCR expression, as evidenced by the fact that disruption of the siteby Cas9, in the absence of a donor template, results in TCR knockout in70% of T cells (see, Eyquem, Supplemental FIG. 1C, second panel).

Notably, nucleases and CRISPR systems disclosed in the prior art eachtarget recognition sequences in T cell receptor genes at sites or locithat are critical for expression of the gene and formation of afunctional T cell receptor; e.g., TRAC exons, or the endogenous spliceacceptor site. Although insertion of a CAR coding sequence into thesecleavage sites can result in T cell receptor negative (TCR−) cells whichare CAR positive (CAR+), a significant disadvantage to this approach isthat TCR expression can be knocked out by error-prone non-homologousend-joining (NHEJ) at the cleavage site when no donor template isinserted.

As a result, previous methods for producing CAR T cells result in mixedpopulations of TCR−/CAR+ and TCR−/CAR− cells that would require furtherenrichment for pre-clinical and clinical use. For example, as previouslydiscussed, Supplemental FIG. 1C of Eyquem shows that ˜70% of cells wereTCR−/CAR− when Cas9 disrupted the splice acceptor site 5′ upstream ofTRAC exon 1 when no donor template is present. However, even in thepresence of a CAR donor template (1e6 AAV6), mixed populations ofTCR−/CAR- and TCR−/CAR+ cells were produced. Specifically, when templateDNA was provided by an AAV6 MOI of 1e6, 45.6% of T cells were TCR−/CAR+,but a substantial percentage of cells (30.7%) were TCR−/CAR− due tocleavage within the splice acceptor site by Cas9 and subsequenterror-prone repair by NHEJ.

By contrast, the present invention takes a counter-intuitive approachfor modifying T genes and inserting sequences of interest, such as CARcoding sequences. Rather than targeting elements of TCR genes that areessential for TCR expression, the present invention targets the intronin the TCR alpha gene that is 5′ upstream of TRAC exon 1. As long as theendogenous splice donor site and the endogenous splice acceptor sitewhich flank the intron are not modified, double-strand cleavage by anuclease within this non-coding intron would have no substantive effecton TCR expression, even if NHEJ produced an indel at the cleavage site.

By going against convention and targeting recognition sequences in theintron, TCR expression is only disrupted when a sequence of interest,comprising at least an exogenous splice acceptor site and/or a poly Asignal, is inserted into the cleavage site, for example, by homologousrecombination. As a result, TCR− cells produced according to theinvention will comprise the sequence of interest inserted into theintron cleavage site. By extension, in cases where the inserted sequenceof interest further includes a CAR coding sequence, most or all of theTCR− cells in the resulting population of cells will be TCR−/CAR+, whichstands in stark contrast to previous methods in which the resultingpopulation would also include a substantial percentage of cells that areTRC−/CAR−. Thus, the invention significantly advances the field byeliminating the burdensome need for enrichment of CAR+ cells from amixed population of TRC− cells.

Further, in some embodiments of the invention, the sequence of interestinserted into the intron comprises a 2A element (see, FIG. 1) 5′upstream of a coding sequence (e.g., a CAR coding sequence). Theinclusion of this 2A element allows for expression of the codingsequence to be driven by the endogenous T cell receptor alpha genepromoter, rather than by an exogenous promoter. In this manner,expression of a polypeptide such as a CAR can be regulated by the T cellfeedback mechanisms normally associated with TCR expression.

SUMMARY OF THE INVENTION

The present invention provides a genetically-modified human T cell, or acell derived therefrom, comprising in its genome a modified human T cellreceptor alpha gene. The modified human T cell receptor alpha gene cancomprise an exogenous sequence of interest inserted into an intronwithin the T cell receptor alpha gene that is positioned 5′ upstream ofTRAC exon 1. The exogenous sequence of interest inserted into the introncan comprise an exogenous splice acceptor site and/or a poly A signal,which disrupts expression of the T cell receptor alpha subunit. In someembodiments, the sequence of interest can also include a coding sequencefor a polypeptide (e.g., a CAR coding sequence). Additionally, theendogenous splice donor site and the endogenous splice acceptor siteflanking the intron are unmodified and/or remain functional in the cell.Further, cell surface expression of an endogenous T cell receptor isreduced when compared to an unmodified control cell.

The present invention also provides compositions and methods forproducing the genetically-modified T cell, as well as populations of Tcells. The present invention further provides a method of immunotherapyfor treating cancer by administering the genetically-modified T cell,wherein the T cell expresses a receptor for a tumor-specific antigen(e.g. a CAR).

Thus, in one aspect, the invention provides an engineered meganucleasethat recognizes and cleaves a recognition sequence within an intron inthe human T cell receptor alpha gene that is positioned 5′ upstream ofTRAC exon 1, wherein the engineered meganuclease comprises a firstsubunit and a second subunit, wherein the first subunit binds to a firstrecognition half-site of the recognition sequence and comprises a firsthypervariable (HVR1) region, and wherein the second subunit binds to asecond recognition half-site of the recognition sequence and comprises asecond hypervariable (HVR2) region. In some embodiments, the introncomprises SEQ ID NO: 3, and the engineered meganuclease does not have arecognition sequence within the endogenous splice donor site or theendogenous splice acceptor site flanking said intron.

In certain embodiments, the recognition sequence comprises SEQ ID NO: 4(i.e., the TRC 11-12 recognition sequence).

In some such embodiments, the HVR1 region comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to an amino acid sequence corresponding toresidues 215-270 of any one of SEQ ID NOs: 12-15.

In some such embodiments, the HVR1 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 12-15.

In some such embodiments, the HVR1 region comprises residues 215-270 ofany one of SEQ ID NOs: 12-15.

In some such embodiments, the HVR2 region comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to an amino acid sequence corresponding toresidues 24-79 of any one of SEQ ID NOs: 12-15.

In some such embodiments, the HVR2 region comprises residuescorresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46,68, 70, 75, and 77 of any one of SEQ ID NOs: 12-15.

In some such embodiments, the HVR2 region comprises residues 24-79 ofany one of SEQ ID NOs: 12-15.

In some such embodiments, the first subunit comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to residues 198-344 of any one of SEQ ID NOs:12-15, and wherein the second subunit comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to residues 7-153 of any one of SEQ ID NOs: 12-15.

In some such embodiments, the first subunit comprises residues 198-344of any one of SEQ ID NOs: 12-15. In some such embodiments, the secondsubunit comprises residues 7-153 of any one of SEQ ID NOs: 12-15.

In some such embodiments, the engineered meganuclease comprises alinker, wherein the linker covalently joins the first subunit and thesecond subunit.

In some such embodiments, the engineered meganuclease comprises theamino acid sequence of any one of SEQ ID NOs: 12-15.

In certain embodiments, the recognition sequence comprises SEQ ID NO: 6(i.e., the TRC 15-16 recognition sequence).

In some such embodiments, the HVR1 region comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to an amino acid sequence corresponding toresidues 24-79 of any one of SEQ ID NOs: 16-19.

In some such embodiments, the HVR1 region comprises residuescorresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46,68, 70, 75, and 77 of any one of SEQ ID NOs: 16-19.

In some such embodiments, the HVR1 region comprises a residuecorresponding to residue 64 of any one of SEQ ID NOs: 16-19.

In some such embodiments, the HVR1 region comprises residues 24-79 ofany one of SEQ ID NOs: 16-19.

In some such embodiments, the HVR2 region comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to an amino acid sequence corresponding toresidues 215-270 of any one of SEQ ID NOs: 16-19.

In some such embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 16-19.

In some such embodiments, the HVR2 region comprises residues 215-270 ofany one of SEQ ID NOs: 16-19.

In some such embodiments, the first subunit comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to residues 7-153 of any one of SEQ ID NOs:16-19, and wherein the second subunit comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to residues 198-344 of any one of SEQ ID NOs: 16-19.

In some such embodiments, the first subunit comprises residues 7-153 ofany one of SEQ ID NOs: 16-19.

In some such embodiments, the second subunit comprises residues 198-344of any one of SEQ ID NOs: 16-19.

In some such embodiments, the engineered meganuclease comprises alinker, wherein the linker covalently joins the first subunit and thesecond subunit.

In some such embodiments, the engineered meganuclease comprises theamino acid sequence of any one of SEQ ID NOs: 16-19.

In certain embodiments, the recognition sequence comprises SEQ ID NO: 8(i.e., the TRC 17-18 recognition sequence).

In some such embodiments, the HVR1 region comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to an amino acid sequence corresponding toresidues 24-79 of any one of SEQ ID NOs: 20-23.

In some such embodiments, the HVR1 region comprises residuescorresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46,68, 70, 75, and 77 of any one of SEQ ID NOs: 20-23.

In some such embodiments, the HVR1 region comprises a residuecorresponding to residue 66 of any one of SEQ ID NOs: 20-23.

In some such embodiments, the HVR1 region comprises residues 24-79 ofany one of SEQ ID NOs: 20-23.

In some such embodiments, the HVR2 region comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to an amino acid sequence corresponding toresidues 215-270 of any one of SEQ ID NOs: 20-23.

In some such embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 20-23.

In some such embodiments, the HVR2 region comprises residues 215-270 ofany one of SEQ ID NOs: 20-23.

In some such embodiments, the first subunit comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to residues 7-153 of any one of SEQ ID NOs:20-23, and wherein the second subunit comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to residues 198-344 of any one of SEQ ID NOs: 20-23.

In some such embodiments, the first subunit comprises residues 7-153 ofany one of SEQ ID NOs: 20-23.

In some such embodiments, the second subunit comprises residues 198-344of any one of SEQ ID NOs: 20-23.

In some such embodiments, the engineered meganuclease comprises alinker, wherein the linker covalently joins the first subunit and thesecond subunit.

In some such embodiments, the engineered meganuclease comprises theamino acid sequence of any one of SEQ ID NOs: 20-23.

In certain embodiments, the recognition sequence comprises SEQ ID NO: 10(i.e., the TRC 19-20 recognition sequence).

In some such embodiments, the HVR1 region comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to an amino acid sequence corresponding toresidues 24-79 of any one of SEQ ID NOs: 24-27.

In some such embodiments, the HVR1 region comprises residuescorresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46,68, 70, 75, and 77 of any one of SEQ ID NOs: 24-27.

In some such embodiments, the HVR1 region comprises residues 24-79 ofany one of SEQ ID NOs: 24-27.

In some such embodiments, the HVR2 region comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to an amino acid sequence corresponding toresidues 215-270 of any one of SEQ ID NOs: 24-27.

In some such embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 24-27.

In some such embodiments, the HVR2 region comprises residues 215-270 ofany one of SEQ ID NOs: 24-27.

In some such embodiments, the first subunit comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to residues 7-153 of any one of SEQ ID NOs:24-27, and wherein the second subunit comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to residues 198-344 of any one of SEQ ID NOs: 24-27.

In some such embodiments, the first subunit comprises residues 7-153 ofany one of SEQ ID NOs: 24-27.

In some such embodiments, the second subunit comprises residues 198-344of any one of SEQ ID NOs: 24-27.

In some such embodiments, the engineered meganuclease comprises alinker, wherein the linker covalently joins the first subunit and thesecond subunit.

In some such embodiments, the engineered meganuclease comprises theamino acid sequence of any one of SEQ ID NOs: 24-27.

In another aspect, the invention provides a polynucleotide comprising anucleic acid sequence encoding an engineered meganuclease describedherein.

In certain embodiments, the polynucleotide is an mRNA.

In further embodiments, the mRNA is a polycistronic mRNA encoding anengineered meganuclease described herein and at least one additionalpolypeptide or nucleic acid.

In another aspect, the invention provides a recombinant DNA constructcomprising the polynucleotide described herein.

In certain embodiments, the recombinant DNA construct encodes a viralvector. In particular embodiments, the viral vector is an adenoviralvector, a lentiviral vector, a retroviral vector, or an adeno-associatedviral (AAV) vector. In specific embodiments, the viral vector is arecombinant AAV vector.

In another aspect, the invention provides a viral vector comprising thepolynucleotide described herein.

In certain embodiments, the viral vector is an adenoviral vector, alentiviral vector, a retroviral vector, or an AAV vector. In particularembodiments, the viral vector is a recombinant AAV vector.

In another aspect, the invention provides a method for producing agenetically-modified T cell comprising an exogenous sequence of interestinserted into a chromosome of the T cell. The method comprisesintroducing into a T cell one or more nucleic acids including: (a) afirst nucleic acid sequence encoding an engineered meganucleasedescribed herein, wherein the engineered meganuclease is expressed inthe T cell; and (b) a second nucleic acid sequence including thesequence of interest; wherein the engineered meganuclease produces acleavage site in the chromosome at a recognition sequence in an intronin the human T cell receptor alpha gene that is positioned 5′ upstreamof TRAC exon 1; and wherein the sequence of interest is inserted intothe chromosome at the cleavage site; and wherein the sequence ofinterest comprises an exogenous splice acceptor site and/or a poly Asignal; and wherein the endogenous splice donor site and the endogenoussplice acceptor site flanking the intron are unmodified and/or remainfunctional.

In some embodiments of the method, the T cell is a precursor T cell inwhich rearrangement of the V and J segments has not occurred.

In certain embodiments of the method, cell surface expression of anendogenous T cell receptor is reduced when compared to an unmodifiedcontrol cell.

In some embodiments of the method, the intron comprises SEQ ID NO: 3.

In some embodiments of the method, the recognition sequence comprisesSEQ ID NO: 4 and the engineered meganuclease is an engineeredmeganuclease described herein which recognizes and cleaves SEQ ID NO: 4.In some embodiments of the method, the recognition sequence comprisesSEQ ID NO: 6 and the engineered meganuclease is an engineeredmeganuclease described herein which recognizes and cleaves SEQ ID NO: 6.In some embodiments of the method, the recognition sequence comprisesSEQ ID NO: 8 and the engineered meganuclease is an engineeredmeganuclease described herein which recognizes and cleaves SEQ ID NO: 8.In some embodiments of the method, the recognition sequence comprisesSEQ ID NO: 10 and the engineered meganuclease is an engineeredmeganuclease described herein which recognizes and cleaves SEQ ID NO:10.

In certain embodiments of the method, the second nucleic acid sequencefurther comprises sequences homologous to sequences flanking thecleavage site and the sequence of interest is inserted at the cleavagesite by homologous recombination.

In some embodiments of the method, the T cell is a human T cell, or acell derived therefrom.

In various embodiments of the method, the sequence of interestcomprises, from 5′ to 3′, an exogenous splice acceptor site, a 2Aelement or IRES element, a coding sequence for a protein of interest,and a polyA signal. In certain embodiments of the method, the 2A elementis a T2A, a P2A, an E2A, or an F2A element. In particular embodiments ofthe method, the 2A element is a T2A element.

In some embodiments of the method, the sequence of interest furthercomprises an exogenous branch site positioned 5′ upstream of theexogenous splice acceptor site.

In some embodiments of the method, the sequence of interest comprises acoding sequence for a chimeric antigen receptor or an exogenous T cellreceptor. In particular embodiments of the method, the chimeric antigenreceptor or the exogenous T cell receptor comprises an extracellularligand-binding domain having specificity for a tumor-specific antigen.

In some embodiments of the method, at least the first nucleic acidsequence is introduced into the T cell by an mRNA.

In certain embodiments of the method, at least the second nucleic acidsequence is introduced into the T cell by a viral vector. In particularembodiments of the method, the viral vector is an adenoviral vector, alentiviral vector, a retroviral vector, or an AAV vector. In specificembodiments of the method, the viral vector is a recombinant AAV vector.

In another aspect, the invention provides a method for producing agenetically-modified T cell comprising an exogenous sequence of interestinserted into a chromosome of the T cell. The method comprises: (a)introducing an engineered meganuclease described herein into a T cell;and (b) transfecting the T cell with a nucleic acid including thesequence of interest; wherein the engineered meganuclease produces acleavage site in the chromosome at a recognition sequence in an intronin the human T cell receptor alpha gene that is positioned 5′ upstreamof TRAC exon 1; and wherein the sequence of interest is inserted intothe chromosome at the cleavage site; and wherein the sequence ofinterest comprises an exogenous splice acceptor site and/or a poly Asignal; and wherein the endogenous splice donor site and the endogenoussplice acceptor site flanking the intron are unmodified and/or remainfunctional.

In some embodiments of the method, the T cell is a precursor T cell inwhich rearrangement of the V and J segments has not occurred.

In some embodiments of the method, cell surface expression of anendogenous T cell receptor is reduced when compared to an unmodifiedcontrol cell.

In certain embodiments of the method, the intron comprises SEQ ID NO: 3.

In some embodiments of the method, the recognition sequence comprisesSEQ ID NO: 4 and the engineered meganuclease is an engineeredmeganuclease described herein which recognizes and cleaves SEQ ID NO: 4.In some embodiments of the method, the recognition sequence comprisesSEQ ID NO: 6 and the engineered meganuclease is an engineeredmeganuclease described herein which recognizes and cleaves SEQ ID NO: 6.In some embodiments of the method, the recognition sequence comprisesSEQ ID NO: 8 and the engineered meganuclease is an engineeredmeganuclease described herein which recognizes and cleaves SEQ ID NO: 8.In some embodiments of the method, the recognition sequence comprisesSEQ ID NO: 10 and the engineered meganuclease is an engineeredmeganuclease described herein which recognizes and cleaves SEQ ID NO:10.

In certain embodiments of the method, the nucleic acid further comprisessequences homologous to sequences flanking the cleavage site and thesequence of interest is inserted at the cleavage site by homologousrecombination.

In some embodiments of the method, the T cell is a human T cell, or acell derived therefrom.

In certain embodiments of the method, the sequence of interestcomprises, from 5′ to 3′, an exogenous splice acceptor site, a 2Aelement or IRES element, a coding sequence for a protein of interest,and a polyA signal. In particular embodiments of the method, the 2Aelement is a T2A, a P2A, an E2A, or an F2A element. In specificembodiments of the method, the 2A element is a T2A element.

In some embodiments of the method, the sequence of interest furthercomprises an exogenous branch site positioned 5′ upstream of theexogenous splice acceptor site.

In some embodiments of the method, the sequence of interest comprises acoding sequence for a chimeric antigen receptor or an exogenous T cellreceptor. In particular embodiments of the method, the chimeric antigenreceptor or the exogenous T cell receptor comprises an extracellularligand-binding domain having specificity for a tumor-specific antigen.

In certain embodiments of the method, the nucleic acid is introducedinto the T cell by a viral vector. In particular embodiments of themethod, the viral vector is an adenoviral vector, a lentiviral vector, aretroviral vector, or an AAV vector. In specific embodiments of themethod, the viral vector is a recombinant AAV vector.

In another aspect, the invention provides a method for producing agenetically-modified T cell comprising a modified human T cell receptoralpha gene. The method comprises: (a) introducing into a T cell: (i) afirst nucleic acid sequence encoding an engineered nuclease, wherein theengineered nuclease is expressed in the T cell; or (ii) an engineerednuclease protein; and (b) introducing into the cell a second nucleicacid sequence comprising an exogenous sequence of interest; wherein theengineered nuclease produces a cleavage site at a recognition sequencewithin an intron in the human T cell receptor alpha gene that ispositioned 5′ upstream of TRAC exon 1; and wherein the sequence ofinterest is inserted into the human T cell receptor alpha gene at thecleavage site; and wherein the sequence of interest comprises anexogenous splice acceptor site and/or a poly A signal; and wherein theendogenous splice donor site and the endogenous splice acceptor siteflanking the intron are unmodified and/or remain functional.

In some embodiments of the method, the T cell is a precursor T cell inwhich rearrangement of the V and J segments has not occurred.

In some embodiments of the method, cell surface expression of anendogenous T cell receptor is reduced when compared to an unmodifiedcontrol cell.

In certain embodiments of the method, the intron comprises SEQ ID NO: 3.

In some embodiments of the method, the second nucleic acid sequencecomprises from 5′ to 3′: (a) a 5′ homology arm that is homologous to the5′ upstream sequence flanking the cleavage site; (b) the exogenoussequence of interest; and (c) a 3′ homology arm that is homologous tothe 3′ downstream sequence flanking the cleavage site; wherein theexogenous sequence of interest is inserted into the human T cellreceptor alpha gene at the cleavage site by homologous recombination.

In some embodiments of the method, the sequence of interest furthercomprises an exogenous branch site positioned 5′ upstream of theexogenous splice acceptor site.

In certain embodiments of the method, the genetically-modified T cell isa genetically-modified human T cell, or a cell derived therefrom.

In some embodiments of the method, the exogenous sequence of interestcomprises, from 5′ to 3′, an exogenous splice acceptor site, a 2Aelement or IRES element, a coding sequence for a protein of interest,and a polyA signal. In certain embodiments of the method, the 2A elementis a T2A, a P2A, an E2A, or an F2A element. In particular embodiments ofthe method, the 2A element is a T2A element.

In some embodiments of the method, the sequence of interest comprises acoding sequence for a chimeric antigen receptor or an exogenous T cellreceptor. In certain embodiments of the method, the chimeric antigenreceptor or the exogenous T cell receptor comprises an extracellularligand-binding domain having specificity for a tumor-specific antigen.

In some embodiments of the method, at least the first nucleic acidsequence is introduced into the T cell by an mRNA.

In certain embodiments of the method, at least the second nucleic acidsequence is introduced into the T cell by a viral vector. In particularembodiments of the method, the viral vector is an adenoviral vector, alentiviral vector, a retroviral vector, or an adeno-associated viral(AAV) vector. In specific embodiments of the method, the viral vector isa recombinant AAV vector.

In some embodiments of the method, the engineered nuclease is anengineered meganuclease, a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TALEN), a compact TALEN, a CRISPRnuclease, or a megaTAL. In particular embodiments of the method, theengineered nuclease is an engineered meganuclease.

In some embodiments of the method, the engineered meganuclease hasspecificity for a recognition sequence comprising SEQ ID NO: 4. In somesuch embodiments of the method, the engineered meganuclease is anengineered meganuclease described herein which recognizes and cleavesSEQ ID NO: 4.

In some embodiments of the method, the engineered meganuclease hasspecificity for a recognition sequence comprising SEQ ID NO: 6. In somesuch embodiments of the method, the engineered meganuclease is anengineered meganuclease described herein which recognizes and cleavesSEQ ID NO: 6.

In some embodiments of the method, the engineered meganuclease hasspecificity for a recognition sequence comprising SEQ ID NO: 8. In somesuch embodiments of the method, the engineered meganuclease is anengineered meganuclease described herein which recognizes and cleavesSEQ ID NO: 8.

In some embodiments of the method, the engineered meganuclease hasspecificity for a recognition sequence comprising SEQ ID NO: 10. In somesuch embodiments of the method, the engineered meganuclease is anengineered meganuclease described herein which recognizes and cleavesSEQ ID NO: 10.

In another aspect, the invention provides a genetically-modified T cellprepared by any of the methods described herein for producing agenetically-modified T cell.

In another aspect, the invention provides a genetically-modified T cellcomprising in its genome a modified human T cell receptor alpha gene,wherein the modified human T cell receptor alpha gene comprises anexogenous sequence of interest inserted into an intron within the T cellreceptor alpha gene that is positioned 5′ upstream of TRAC exon 1, andwherein the exogenous sequence of interest comprises an exogenous spliceacceptor site and/or a poly A signal, and wherein the endogenous splicedonor site and the endogenous splice acceptor site flanking the intronare unmodified and/or remain functional, and wherein cell surfaceexpression of an endogenous T cell receptor is reduced when compared toan unmodified control cell.

In some embodiments, the intron comprises SEQ ID NO: 3.

In certain embodiments, the genetically-modified T cell is agenetically-modified human T cell, or a cell derived therefrom.

In some embodiments, the exogenous sequence of interest comprises, from5′ to 3′, an exogenous splice acceptor site, a 2A element or IRESelement, a coding sequence for a protein of interest, and a polyAsignal. In particular embodiments, the 2A element is a T2A, a P2A, anE2A, or an F2A element. In specific embodiments, the 2A element is a T2Aelement.

In some embodiments, the exogenous sequence of interest furthercomprises an exogenous branch site positioned 5′ upstream of theexogenous splice acceptor site.

In certain embodiments, the sequence of interest comprises a codingsequence for a chimeric antigen receptor or an exogenous T cellreceptor. In particular embodiments, the chimeric antigen receptor orthe exogenous T cell receptor comprises an extracellular ligand-bindingdomain having specificity for a tumor-specific antigen.

In some embodiments, the exogenous sequence of interest is inserted intothe intron at an engineered meganuclease recognition site, a TALENrecognition site, a zinc finger nuclease recognition site, a CRISPRrecognition site, or a megaTAL recognition site. In particularembodiments, the exogenous sequence of interest is inserted into theintron at an engineered meganuclease recognition site. In specificembodiments, the exogenous sequence of interest is inserted into theintron within SEQ ID NO: 4. In other embodiments, the exogenous sequenceof interest is inserted into the intron within SEQ ID NO: 6. In furtherembodiments, the exogenous sequence of interest is inserted into theintron within SEQ ID NO: 8. In other embodiments, the exogenous sequenceof interest is inserted into the intron within SEQ ID NO: 10.

In another aspect, the invention provides a population ofgenetically-modified T cells comprising a plurality of agenetically-modified T cell described herein.

In some embodiments, at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or up to 100%, of cellsin the population are a genetically-modified T cell as described herein.

In particular embodiments, the genetically-modified T cell is agenetically-modified human T cell, or cell derived therefrom.

In some embodiments, the exogenous sequence of interest present in thegenetically-modified T cell comprises a coding sequence for a chimericantigen receptor or an exogenous T cell receptor. In particularembodiments, the chimeric antigen receptor or the exogenous T cellreceptor comprises an extracellular ligand-binding domain havingspecificity for a tumor-specific antigen.

In some embodiments, cell surface expression of an endogenous T cellreceptor is reduced on the genetically-modified T cell when compared toan unmodified control cell.

In another aspect, the invention provides a pharmaceutical compositionuseful for the treatment of a disease in a subject in need thereof,wherein the pharmaceutical composition comprises apharmaceutically-acceptable carrier and a therapeutically effectiveamount of a genetically-modified T cell as described herein.

In certain embodiments, the genetically-modified T cell is agenetically-modified human T cell, or a cell derived therefrom.

In some embodiments, the exogenous sequence of interest present in thegenetically-modified T cell comprises a coding sequence for a chimericantigen receptor or an exogenous T cell receptor. In certain particularembodiments, the chimeric antigen receptor or the exogenous T cellreceptor comprises an extracellular ligand-binding domain havingspecificity for a tumor-specific antigen.

In some embodiments, cell surface expression of an endogenous T cellreceptor is reduced on the genetically-modified T cell when compared toan unmodified control cell.

In another aspect, the invention provides a method of treating a diseasein a subject in need thereof, the method comprising administering to thesubject a genetically-modified T cell as described herein.

In some embodiments, the method comprises administering to the subject apharmaceutical composition described herein.

In certain embodiments, the method is an immunotherapy for the treatmentof cancer in a subject in need thereof. In some such embodiments, thegenetically-modified T cell is a genetically-modified human T cell, or acell derived therefrom, the exogenous sequence of interest present inthe genetically-modified T cell comprises a coding sequence for achimeric antigen receptor or an exogenous T cell receptor comprising anextracellular ligand-binding domain having specificity for atumor-specific antigen, and cell surface expression of an endogenous Tcell receptor is reduced on the genetically-modified T cell whencompared to an unmodified control cell.

In some embodiments of the method, the cancer is selected from the groupconsisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, andleukemia.

In certain embodiments of the method, the cancer is selected from thegroup consisting of a cancer of B cell origin, breast cancer, gastriccancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostatecancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma.

In particular embodiments of the method, the cancer of B cell origin isselected from the group consisting of B lineage acute lymphoblasticleukemia, B cell chronic lymphocytic leukemia, B cell non-Hodgkin'slymphoma, and multiple myeloma.

In another aspect, the invention provides a genetically-modified cell,as described herein, for use as a medicament. The invention furtherprovides the use of a genetically-modified cell, as described herein, inthe manufacture of a medicament for treating a disease in a subject inneed thereof. In one such aspect, the medicament is useful in thetreatment of cancer.

In another aspect, the invention provides a genetically-modified cell,as described herein, for use in treatment of a disease, and preferablyin the treatment of cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Diagram of sample strategy for insertion and expression of anexogenous sequence of interest into an intron of a T cell receptor alphagene, which has been rearranged to encode a functional T cell receptoralpha subunit. As shown, an exogenous sequence of interest is insertedinto the intron of the T cell receptor alpha gene that is 5′ upstream ofTRAC exon 1. The endogenous splice acceptor site and the endogenoussplice acceptor site which flank the targeted 5′ intron remain intact.Following cleavage by a nuclease, an exogenous sequence of interestdescribed herein is inserted into the intron. As shown, the sequence ofinterest comprises at least an exogenous splice acceptor site and/or apoly A signal which, when inserted into the intron, will disruptexpression of the T cell receptor alpha subunit. The inserted sequenceof interest can optionally include a 2A element, which is represented bya T2A element. The inserted sequence of interest can also optionallyinclude a coding sequence for a polypeptide of interest, which isrepresented by a chimeric antigen receptor coding sequence. Ifnecessary, the sequence of interest can further comprise an exogenousbranch site positioned 5′ upstream of the exogenous splice acceptorsite.

FIG. 2. TRC recognition sequences in the targeted 5′ intron of the humanT cell receptor alpha gene. Each recognition sequence targeted by anengineered meganuclease of the invention comprises two recognitionhalf-sites. Each recognition half-site comprises 9 base pairs, separatedby a 4 base pair central sequence. The TRC 11-12 recognition sequence(SEQ ID NO: 4) comprises two recognition half-sites referred to as TRC11and TRC12. The TRC 15-16 recognition sequence (SEQ ID NO: 6) comprisestwo recognition half-sites referred to as TRC15 and TRC16. The TRC 17-18recognition sequence (SEQ ID NO: 8) comprises two recognition half-sitesreferred to as TRC17 and TRC18. The TRC 19-20 recognition sequence (SEQID NO: 10) comprises two recognition half-sites referred to as TRC19 andTRC20.

FIG. 3. The engineered meganucleases of the invention comprise twosubunits, wherein the first subunit comprising the HVR1 region binds toa first recognition half-site (e.g., TRC11, TRC15, TRC17, or TRC19) andthe second subunit comprising the HVR2 region binds to a secondrecognition half-site (e.g., TRC12, TRC16, TRC18, or TRC20). Inembodiments where the engineered meganuclease is a single-chainmeganuclease, the first subunit comprising the HVR1 region can bepositioned as either the N-terminal or C-terminal subunit. Likewise, thesecond subunit comprising the HVR2 region can be positioned as eitherthe N-terminal or C-terminal subunit.

FIG. 4. Schematic of reporter assay in CHO cells for evaluatingengineered meganucleases targeting recognition sequences found in thetargeted 5′ intron of the T cell receptor alpha gene. For the engineeredmeganucleases described herein, a CHO cell line was produced in which areporter cassette was integrated stably into the genome of the cell. Thereporter cassette comprised, in 5′ to 3′ order: an SV40 Early Promoter;the 5′ 2/3 of the GFP gene; the recognition sequence for an engineeredmeganuclease of the invention (e.g., the TRC 11-12 recognitionsequence); the recognition sequence for the CHO-23/24 meganuclease(WO/2012/167192); and the 3′ 2/3 of the GFP gene. Cells stablytransfected with this cassette did not express GFP in the absence of aDNA break-inducing agent. Meganucleases were introduced by transductionof plasmid DNA or mRNA encoding each meganuclease. When a DNA break wasinduced at either of the meganuclease recognition sequences, theduplicated regions of the GFP gene recombined with one another toproduce a functional GFP gene. The percentage of GFP-expressing cellscould then be determined by flow cytometry as an indirect measure of thefrequency of genome cleavage by the engineered meganucleases.

FIGS. 5A-5D. Efficiency of engineered meganucleases for recognizing andcleaving recognition sequences in the found in the targeted 5′ intron ofthe T cell receptor alpha gene in a CHO cell reporter assay. Engineeredmeganucleases set forth in SEQ ID NOs: 12-15 were engineered to targetthe TRC 11-12 recognition sequence (SEQ ID NO: 4). Engineeredmeganucleases set forth in SEQ ID NOs: 16-19 were engineered to targetthe TRC 15-16 recognition sequence (SEQ ID NO: 6), and were screened forefficacy in the CHO cell reporter assay. Engineered meganucleases setforth in SEQ ID NOs: 20-23 were engineered to target the TRC 17-18recognition sequence (SEQ ID NO: 8). Engineered meganucleases set forthin SEQ ID NOs: 24-27 were engineered to target the TRC 19-20 recognitionsequence (SEQ ID NO: 10). The results shown provide the percentage ofGFP-expressing cells observed in each assay, which indicates theefficacy of each meganuclease for cleaving a target recognition sequenceor the CHO-23/24 recognition sequence. A negative control (bs) wasfurther included in each assay. FIG. 5A shows meganucleases targetingthe TRC 11-12 recognition sequence. FIG. 5B shows meganucleasestargeting the TRC 15-16 recognition sequence. FIG. 5C showsmeganucleases targeting the TRC 17-18 recognition sequence. FIG. 5Dshows meganucleases targeting the TRC 19-20 recognition sequence.

FIGS. 6A-6D. Efficiency of engineered meganucleases for recognizing andcleaving recognition sequences in the intron of the human T cellreceptor alpha gene which is 5′ upstream of TRAC exon 1 in a CHO cellreporter assay. Engineered meganucleases set forth in SEQ ID NOs: 12-15were engineered to target the TRC 11-12 recognition sequence (SEQ ID NO:4). Engineered meganucleases set forth in SEQ ID NOs: 16-19 wereengineered to target the TRC 15-16 recognition sequence (SEQ ID NO: 6)and were screened for efficacy in the CHO cell reporter assay.Engineered meganucleases set forth in SEQ ID NOs: 20-23 were engineeredto target the TRC 17-18 recognition sequence (SEQ ID NO: 8). Engineeredmeganucleases set forth in SEQ ID NOs: 24-27 were engineered to targetTRC 19-20 recognition sequence (SEQ ID NO: 10). The engineeredmeganucleases were screened for efficacy in the CHO cell reporter assayat multiple time points over 7 days after nucleofection. The resultsshown provide the percentage of GFP-expressing cells observed in eachassay over the 7-day period of analysis, which indicates the efficacy ofeach meganuclease for cleaving a target recognition sequence or theCHO-23/24 recognition sequence as a function of time. FIG. 6A showsmeganucleases targeting the TRC 11-12 recognition sequence. FIG. 6Bshows meganucleases targeting the TRC 15-16 recognition sequence. FIG.6C shows meganucleases targeting the TRC 17-18 recognition sequence.FIG. 6D shows meganucleases targeting the TRC 19-20 recognitionsequence.

FIG. 7. T7E assay of T cell lysates. Human CD3+ T cells were isolatedfrom PBMCs by magnetic separation and activated for 72 hours. Activatedhuman T cells were electroporated with TRC 11-12 or TRC 15-16meganuclease mRNA and, at 72 hours post-transfection, genomic DNA (gDNA)was harvested from cells. A T7 endonuclease I (T7E) assay was performedto estimate genetic modification at the endogenous TRC 11-12 or TRC15-16 recognition sequence.

FIG. 8. Cleavage at recognition sequences in the targeted 5′ intron donot affect T cell receptor expression. Human T cells were enriched froman apheresis sample obtained from a human donor and were stimulated for3 days using antiCD3/antiCD28 beads in the presence of IL-2. After 3days, T cells were harvested, beads were removed, and 1 μg of theindicated meganuclease RNA was introduced to T cell samples.Nucleofected cells were cultured for 6 days prior to flow cytometricanalysis. CD3 surface display, representative of endogenous T cellreceptor expression, was measured by labeling T cell samples withanti-CD3-BrilliantViolet711 and GhostDye-510. T cells were nucleofectedwith either TRC 1-2x.87EE (an engineered nuclease which targets TRACexon 1) or no RNA (mock) to serve as positive and negative controls forTRAC locus editing, respectively, and appear in FIG. 8A and FIG. 8B.Four additional samples were also nucleofected with RNA encoding onedistinct nuclease variant from the TRC 15-16 family, all members ofwhich target the TRC 15-16 recognition sequence in the 5′ intron. WhenTRAC locus editing results in gene disruption, no TCRα chains aresynthesized, and no TCR complex (including CD3) is displayed on thesurface of edited cells. Greater than half of the TRC 1-2x.87EE edited Tcells were shown to be TRC negative due to cleavage in exon 1 anderror-prone repair of the cleavage cite by NHEJ (FIG. 8B). Bycomparison, the frequency of TCR negative cells following editing by TRC15-16x.31, TRC 15-16x.63, TRC 15-16x.87, and TRC 15-16x.89 was betweenonly 4% and 8% (FIGS. 8C, 8D, 8E, and 8F, respectively).

FIGS. 9A-9C. Donor templates for exogenous sequence of interest. Donortemplates comprising homology arms, an exogenous splice acceptor site, aCAR coding sequence, and a poly A signal, are provided. FIG. 9A providesan example donor template (SEQ ID NO: 60) suitable for insertion intothe TRC 11-12 recognition sequence. FIG. 9B provides an example donortemplate (SEQ ID NO: 61) suitable for insertion into the TRC 15-16recognition sequence. FIG. 9C provides an example donor template (SEQ IDNO: 62) suitable for insertion into the TRC 17-18 recognition sequence.

FIG. 10. Insertion of a GFP coding sequence into the targeted 5′ intron.T cells were nucleofected with mRNA encoding the TRC 11-12x.82 nucleaseand were transduced with an AAV6 vector comprising the 7227 construct,which encodes a T2A sequence followed by a promoterless GFP codingsequence. Additional T cells were nucleofected with mRNA encoding theTRC 15-16.x31 nuclease and were transduced with an AAV6 vectorcomprising the 7228 construct, which encodes a T2A sequence followed bya promoterless GFP coding sequence. TCR knockout and GFP expression weredetermined by flow cytometry 5 days after transfection/transduction.FIG. 10A shows CD3 (x-axis) and GFP (y-axis) expression following donortemplate insertion at the TRC 11-12 recognition sequence. FIG. 10B showsGFP expression (x-axis) and cell count (y-axis) following donor templateinsertion at the TRC 11-12 recognition sequence. FIG. 10C shows CD3(x-axis) and GFP (y-axis) expression following donor template insertionat the TRC 15-16 recognition sequence. FIG. 10D shows GFP expression(x-axis) and cell count (y-axis) following donor template insertion atthe TRC 15-16 recognition sequence.

FIG. 11. Insertion of an anti-CD19 CAR coding sequence into the targeted5′ intron. T cells were nucleofected with mRNA encoding the TRC11-12x.82 nuclease and were transduced with an AAV6 vector comprisingthe 7225 construct, which encodes a T2A sequence followed by apromoterless anti-CD19 CAR coding sequence. Additional T cells werenucleofected with mRNA encoding the TRC 15-16.x31 nuclease and weretransduced with an AAV6 vector comprising the 7226 construct, whichencodes a T2A sequence followed by a promoterless anti-CD19 CAR codingsequence. TCR knockout and CAR expression were determined by flowcytometry 5 days after transfection/transduction. FIG. 11A shows CARexpression on CD3− cells in a negative control group (TRC enzyme only).

FIG. 11B shows CAR expression on CD3− cells following donor templateinsertion at the TRC 11-12 recognition sequence. FIG. 11C shows CARexpression on CD3− cells following donor template insertion at the TRC15-16 recognition sequence.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth the amino acid sequence of wild-type I-CreImeganuclease.

SEQ ID NO: 2 sets forth the amino acid sequence of LAGLIDADG.

SEQ ID NO: 3 sets forth a nucleic acid sequence of the human T cellreceptor alpha gene intron.

SEQ ID NO: 4 sets forth the nucleic acid sequence of TRC 11-12 (sense).

SEQ ID NO: 5 sets forth the nucleic acid sequence of TRC 11-12(antisense).

SEQ ID NO: 6 sets forth the nucleic acid sequence of TRC 15-16 (sense).

SEQ ID NO: 7 sets forth the nucleic acid sequence of TRC 15-16(antisense).

SEQ ID NO: 8 sets forth the nucleic acid sequence of TRC 17-18 (sense).

SEQ ID NO: 9 sets forth the nucleic acid sequence of TRC 17-18(antisense).

SEQ ID NO: 10 sets forth the nucleic acid sequence of TRC 19-20 (sense).

SEQ ID NO: 11 sets forth the nucleic acid sequence of TRC 19-20(antisense).

SEQ ID NO: 12 sets forth the amino acid sequence of the TRC 11-12x.4meganuclease.

SEQ ID NO: 13 sets forth the amino acid sequence of the TRC 11-12x.82meganuclease.

SEQ ID NO: 14 sets forth the amino acid sequence of the TRC 11-12x.60meganuclease.

SEQ ID NO: 15 sets forth the amino acid sequence of the TRC 11-12x.63meganuclease.

SEQ ID NO: 16 sets forth the amino acid sequence of the TRC 15-16x.31meganuclease.

SEQ ID NO: 17 sets forth the amino acid sequence of the TRC 15-16x.87meganuclease.

SEQ ID NO: 18 sets forth the amino acid sequence of the TRC 15-16x.63meganuclease.

SEQ ID NO: 19 sets forth the amino acid sequence of the TRC 15-16x.89meganuclease.

SEQ ID NO: 20 sets forth the amino acid sequence of the TRC17-18x.15meganuclease.

SEQ ID NO: 21 sets forth the amino acid sequence of the TRC17-18x.82meganuclease.

SEQ ID NO: 22 sets forth the amino acid sequence of the TRC17-18x.18meganuclease.

SEQ ID NO: 23 sets forth the amino acid sequence of the TRC17-18x.71meganuclease.

SEQ ID NO: 24 sets forth the amino acid sequence of the TRC 19-20x.85meganuclease.

SEQ ID NO: 25 sets forth the amino acid sequence of the TRC 19-20x.74meganuclease.

SEQ ID NO: 26 sets forth the amino acid sequence of the TRC 19-20x.71meganuclease.

SEQ ID NO: 27 sets forth the amino acid sequence of the TRC 19-20x.87meganuclease.

SEQ ID NO: 28 sets forth the amino acid sequence of the TRC 11-12x.4meganuclease TRC11-binding subunit.

SEQ ID NO: 29 sets forth the amino acid sequence of the TRC 11-12x.82meganuclease TRC11-binding subunit.

SEQ ID NO: 30 sets forth the amino acid sequence of the TRC 11-12x.60meganuclease TRC11-binding subunit.

SEQ ID NO: 31 sets forth the amino acid sequence of the TRC 11-12x.63meganuclease TRC11-binding subunit.

SEQ ID NO: 32 sets forth the amino acid sequence of the TRC 11-12x.4meganuclease TRC12-binding subunit.

SEQ ID NO: 33 sets forth the amino acid sequence of the TRC 11-12x.82meganuclease TRC12-binding subunit.

SEQ ID NO: 34 sets forth the amino acid sequence of the TRC 11-12x.60meganuclease TRC12-binding subunit.

SEQ ID NO: 35 sets forth the amino acid sequence of the TRC 11-12x.63meganuclease TRC12-binding subunit.

SEQ ID NO: 36 sets forth the amino acid sequence of the TRC 15-16x.31meganuclease TRC15-binding subunit.

SEQ ID NO: 37 sets forth the amino acid sequence of the TRC 15-16x.87meganuclease TRC15-binding subunit.

SEQ ID NO: 38 sets forth the amino acid sequence of the TRC 15-16x.63meganuclease TRC15-binding subunit.

SEQ ID NO: 39 sets forth the amino acid sequence of the TRC 15-16x.89meganuclease TRC15-binding subunit.

SEQ ID NO: 40 sets forth the amino acid sequence of the TRC 15-16x.31meganuclease TRC16-binding subunit.

SEQ ID NO: 41 sets forth the amino acid sequence of the TRC 15-16x.87meganuclease TRC16-binding subunit.

SEQ ID NO: 42 sets forth the amino acid sequence of the TRC 15-16x.63meganuclease TRC16-binding subunit.

SEQ ID NO: 43 sets forth the amino acid sequence of the TRC 15-16x.89meganuclease TRC16-binding subunit.

SEQ ID NO: 44 sets forth the amino acid sequence of the TRC17-18x.15meganuclease TRC17-binding subunit.

SEQ ID NO: 45 sets forth the amino acid sequence of the TRC17-18x.82meganuclease TRC17-binding subunit.

SEQ ID NO: 46 sets forth the amino acid sequence of the TRC17-18x.18meganuclease TRC17-binding subunit.

SEQ ID NO: 47 sets forth the amino acid sequence of the TRC17-18x.71meganuclease TRC17-binding subunit.

SEQ ID NO: 48 sets forth the amino acid sequence of the TRC17-18x.15meganuclease TRC18-binding subunit.

SEQ ID NO: 49 sets forth the amino acid sequence of the TRC17-18x.82meganuclease TRC18-binding subunit.

SEQ ID NO: 50 sets forth the amino acid sequence of the TRC17-18x.18meganuclease TRC18-binding subunit.

SEQ ID NO: 51 sets forth the amino acid sequence of the TRC17-18x.71meganuclease TRC18-binding subunit.

SEQ ID NO: 52 sets forth the amino acid sequence of the TRC 19-20x.85meganuclease TRC19-binding subunit.

SEQ ID NO: 53 sets forth the amino acid sequence of the TRC 19-20x.74meganuclease TRC19-binding subunit.

SEQ ID NO: 54 sets forth the amino acid sequence of the TRC 19-20x.71meganuclease TRC19-binding subunit.

SEQ ID NO: 55 sets forth the amino acid sequence of the TRC 19-20x.87meganuclease TRC19-binding subunit.

SEQ ID NO: 56 sets forth the amino acid sequence of the TRC 19-20x.85meganuclease TRC20-binding subunit.

SEQ ID NO: 57 sets forth the amino acid sequence of the TRC 19-20x.74meganuclease TRC20-binding subunit.

SEQ ID NO: 58 sets forth the amino acid sequence of the TRC 19-20x.71meganuclease TRC20-binding subunit.

SEQ ID NO: 59 sets forth the amino acid sequence of the TRC 19-20x.87meganuclease TRC20-binding subunit.

SEQ ID NO: 60 sets forth the nucleic acid sequence of a donor templatecomprising an anti-CD19 CAR that can be inserted at the TRC 11-12recognition sequence.

SEQ ID NO: 61 sets forth the nucleic acid sequence of a donor templatecomprising an anti-CD19 CAR that can be inserted at the TRC 15-16recognition sequence.

SEQ ID NO: 62 sets forth the nucleic acid sequence of a donor templatecomprising an anti-CD19 CAR that can be inserted at the TRC 17-18recognition sequence.

SEQ ID NO: 63 sets forth the nucleic acid sequence of the 7227 donortemplate encoding a GFP protein that can be inserted at the TRC 11-12recognition sequence.

SEQ ID NO: 64 sets forth the nucleic acid sequence of the 7225 donortemplate encoding an anti-CD19 CAR that can be inserted at the TRC 11-12recognition sequence.

SEQ ID NO: 65 sets forth the nucleic acid sequence of the 7228 donortemplate encoding a GFP protein that can be inserted at the TRC 15-16recognition sequence.

SEQ ID NO: 66 sets forth the nucleic acid sequence of the 7226 donortemplate encoding an anti-CD19 CAR that can be inserted at the TRC 15-16recognition sequence.

DETAILED DESCRIPTION OF THE INVENTION 1.1 References and Definitions

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The issued USpatents, allowed applications, published foreign applications, andreferences, including GenBank database sequences, which are cited hereinare hereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

The present invention can be embodied in different forms and should notbe construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference herein in their entirety.

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

As used herein, the term “meganuclease” refers to an endonuclease thatbinds double-stranded DNA at a recognition sequence that is greater than12 base pairs. Preferably, the recognition sequence for a meganucleaseof the invention is 22 base pairs. A meganuclease can be an endonucleasethat is derived from I-CreI, and can refer to an engineered variant ofI-CreI that has been modified relative to natural I-CreI with respectto, for example, DNA-binding specificity, DNA cleavage activity,DNA-binding affinity, or dimerization properties. Methods for producingsuch modified variants of I-CreI are known in the art (e.g., WO2007/047859). A meganuclease as used herein binds to double-stranded DNAas a heterodimer or as a “single-chain meganuclease” in which a pair ofDNA-binding domains are joined into a single polypeptide using a peptidelinker. The term “homing endonuclease” is synonymous with the term“meganuclease.” Meganucleases of the invention are substantiallynon-toxic when expressed in cells, particularly in human T cells, suchthat cells can be transfected and maintained at 37° C. without observingdeleterious effects on cell viability or significant reductions inmeganuclease cleavage activity when measured using the methods describedherein.

As used herein, the term “single-chain meganuclease” refers to apolypeptide comprising a pair of nuclease subunits joined by a linker. Asingle-chain meganuclease has the organization: N-terminalsubunit-Linker-C-terminal subunit. The two meganuclease subunits willgenerally be non-identical in amino acid sequence and will recognizenon-identical DNA sequences. Thus, single-chain meganucleases typicallycleave pseudo-palindromic or non-palindromic recognition sequences. Asingle-chain meganuclease may be referred to as a “single-chainheterodimer” or “single-chain heterodimeric meganuclease” although it isnot, in fact, dimeric. For clarity, unless otherwise specified, the term“meganuclease” can refer to a dimeric or single-chain meganuclease.

As used herein, the term “linker” refers to an exogenous peptidesequence used to join two meganuclease subunits into a singlepolypeptide. A linker may have a sequence that is found in naturalproteins, or may be an artificial sequence that is not found in anynatural protein. A linker may be flexible and lacking in secondarystructure or may have a propensity to form a specific three-dimensionalstructure under physiological conditions. A linker can include, withoutlimitation, those encompassed by U.S. Pat. Nos. 8,445,251 and 9,434,931.In some embodiments, a linker may have an amino acid sequence comprisingresidues 154-195 of any one of SEQ ID NOs: 12-27.

As used herein, the term “zinc finger nuclease” or “ZFN” refers tochimeric proteins comprising a zinc finger DNA-binding domain fused to anuclease domain from an endonuclease or exonuclease, including but notlimited to a restriction endonuclease, homing endonuclease, 51 nuclease,mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeastHO endonuclease. Nuclease domains useful for the design of zinc fingernuclease include those from a Type IIs restriction endonuclease,including but not limited to FokI, FoM, StsI restriction enzyme.Additional Type IIs restriction endonucleases are described inInternational Publication No. WO 2007/014275, which is incorporated byreference in its entirety. The structure of a zinc finger domain isstabilized through coordination of a zinc ion. DNA binding proteinscomprising one or more zinc finger domains bind DNA in asequence-specific manner. The zinc finger domain can be a nativesequence or can be redesigned through rational or experimental means toproduce a protein which binds to a pre-determined DNA sequence ˜18basepairs in length. See, for example, U.S. Pat. Nos. 5,789,538,5,925,523, 6,007,988, 6,013,453, 6,200,759, and InternationalPublication Nos. WO 95/19431, WO 96/06166, WO 98/53057, WO 98/54311, WO00/27878, WO 01/60970, WO 01/88197, and WO 02/099084, each of which isincorporated by reference in its entirety. By fusing this engineeredprotein domain to a nuclease domain, such as FokI nuclease, it ispossible to target DNA breaks with genome-level specificity. Theselection of target sites, zinc finger proteins and methods for designand construction of zinc finger nucleases are known to those of skill inthe art and are described in detail in U.S. Publications Nos.20030232410, 20050208489, 2005064474, 20050026157, 20060188987 andInternational Publication No. WO 07/014275, each of which isincorporated by reference in its entirety.

As used herein, the term “TALEN” refers to an endonuclease comprising aDNA-binding domain comprising a plurality of TAL domain repeats fused toa nuclease domain or an active portion thereof from an endonuclease orexonuclease, including but not limited to a restriction endonuclease,homing endonuclease, 51 nuclease, mung bean nuclease, pancreatic DNAseI, micrococcal nuclease, and yeast HO endonuclease. See, for example,Christian et al. (2010) Genetics 186:757-761, which is incorporated byreference in its entirety. Nuclease domains useful for the design ofTALENs include those from a Type IIs restriction endonuclease, includingbut not limited to FokI, FoM, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI,BglI, and AlwI. Additional Type IIs restriction endonucleases aredescribed in International Publication No. WO 2007/014275. In someembodiments, the nuclease domain of the TALEN is a FokI nuclease domainor an active portion thereof. TAL domain repeats can be derived from theTALE (transcription activator-like effector) family of proteins used inthe infection process by plant pathogens of the Xanthomonas genus. TALdomain repeats are 33-34 amino acid sequences with divergent 12^(th) and13^(th) amino acids. These two positions, referred to as the repeatvariable dipeptide (RVD), are highly variable and show a strongcorrelation with specific nucleotide recognition. Each base pair in theDNA target sequence is contacted by a single TAL repeat, with thespecificity resulting from the RVD. In some embodiments, the TALENcomprises 16-22 TAL domain repeats. DNA cleavage by a TALEN requires twoDNA recognition regions flanking a nonspecific central region (i.e., the“spacer”). The term “spacer” in reference to a TALEN refers to thenucleic acid sequence that separates the two nucleic acid sequencesrecognized and bound by each monomer constituting a TALEN. The TALdomain repeats can be native sequences from a naturally-occurring TALEprotein or can be redesigned through rational or experimental means toproduce a protein which binds to a pre-determined DNA sequence (see, forexample, Boch et al. (2009) Science 326(5959):1509-1512 and Moscou andBogdanove (2009) Science 326(5959):1501, each of which is incorporatedby reference in its entirety). See also, U.S. Publication No.20110145940 and International Publication No. WO 2010/079430 for methodsfor engineering a TALEN to recognize a specific sequence and examples ofRVDs and their corresponding target nucleotides. In some embodiments,each nuclease (e.g., FokI) monomer can be fused to a TAL effectorsequence that recognizes a different DNA sequence, and only when the tworecognition sites are in close proximity do the inactive monomers cometogether to create a functional enzyme.

As used herein, the term “Compact TALEN” refers to an endonucleasecomprising a DNA-binding domain with 16-22 TAL domain repeats fused inany orientation to any portion of the I-TevI homing endonuclease or anyof the endonucleases listed in Table 2 in U.S. Application No.20130117869 (which is incorporated by reference in its entirety),including but not limited to MmeI, EndA, End1, I-BasI, I-TevII,I-TevIII, I-TwoI, MspI, MvaI, NucA, and NucM. Compact TALENs do notrequire dimerization for DNA processing activity, alleviating the needfor dual target sites with intervening DNA spacers. In some embodiments,the compact TALEN comprises 16-22 TAL domain repeats.

As used herein, the term “CRISPR” refers to a Cas-based endonucleasecomprising a Cas, such as Cas9, Cpf1, or another suitable nuclease, anda guide RNA that directs DNA cleavage of the Cas by hybridizing to arecognition site in the genomic DNA. The Cas component of a CRISPR is anRNA-guided DNA endonuclease. In certain embodiments, the Cas is a classII Cas enzyme. In some of these embodiments, the Cas is a class II, typeII enzyme, such as Cas9. In other embodiments, the Cas is a class II,type V enzyme, such as Cpf1. The guide RNA comprises a direct repeat anda guide sequence (often referred to as a spacer in the context of anendogenous CRISPR system), which is complementary to the targetrecognition site. In certain embodiments, the CRISPR further comprises atracrRNA (trans-activating CRISPR RNA) that is complementary (fully orpartially) to a direct repeat sequence (sometimes referred to as atracr-mate sequence) present on the guide RNA. In particularembodiments, the Cas can be mutated with respect to a correspondingwild-type enzyme such that the enzyme lacks the ability to cleave onestrand of a target polynucleotide, functioning as a nickase, cleavingonly a single strand of the target DNA. Non-limiting examples of Casenzymes that function as a nickase include Cas9 enzymes with a D10Amutation within the RuvC I catalytic domain, or with a H840A, N854A, orN863A mutation.

As used herein, the term “megaTAL” refers to a single-chain nucleasecomprising a transcription activator-like effector (TALE) DNA bindingdomain with an engineered, sequence-specific homing endonuclease.

As used herein, with respect to a protein, the terms “recombinant” or“engineered” means having an altered amino acid sequence as a result ofthe application of genetic engineering techniques to nucleic acids thatencode the protein, and cells or organisms that express the protein.With respect to a nucleic acid, the term “recombinant” or “engineered”means having an altered nucleic acid sequence as a result of theapplication of genetic engineering techniques. Genetic engineeringtechniques include, but are not limited to, PCR and DNA cloningtechnologies; transfection, transformation and other gene transfertechnologies; homologous recombination; site-directed mutagenesis; andgene fusion. In accordance with this definition, a protein having anamino acid sequence identical to a naturally-occurring protein, butproduced by cloning and expression in a heterologous host, is notconsidered recombinant or engineered.

As used herein, the term “wild-type” refers to the most common naturallyoccurring allele (i.e., polynucleotide sequence) in the allelepopulation of the same type of gene, wherein a polypeptide encoded bythe wild-type allele has its original functions. The term “wild-type”also refers a polypeptide encoded by a wild-type allele. Wild-typealleles (i.e., polynucleotides) and polypeptides are distinguishablefrom mutant or variant alleles and polypeptides, which comprise one ormore mutations and/or substitutions relative to the wild-typesequence(s). Whereas a wild-type allele or polypeptide can confer anormal phenotype in an organism, a mutant or variant allele orpolypeptide can, in some instances, confer an altered phenotype.Wild-type nucleases are distinguishable from recombinant, engineered, ornon-naturally-occurring nucleases.

As used herein with respect to recombinant or engineered proteins, theterm “modification” means any insertion, deletion or substitution of anamino acid residue in the recombinant sequence relative to a referencesequence (e.g., a wild-type or a native sequence).

As used herein, the term “recognition sequence” refers to a DNA sequencethat is bound and cleaved by an endonuclease. In the case of ameganuclease, a recognition sequence comprises a pair of inverted, 9base pair “half sites” which are separated by four basepairs. In thecase of a single-chain meganuclease, the N-terminal domain of theprotein contacts a first half-site and the C-terminal domain of theprotein contacts a second half-site. Cleavage by a meganuclease producesfour base pair 3′ “overhangs”. “Overhangs,” or “sticky ends” are short,single-stranded DNA segments that can be produced by endonucleasecleavage of a double-stranded DNA sequence. In the case of meganucleasesand single-chain meganucleases derived from I-CreI, the overhangcomprises bases 10-13 of the 22 base pair recognition sequence. In thecase of a compact TALEN, the recognition sequence can comprises a firstCNNNGN sequence that is recognized by the I-TevI domain, followed by anon-specific spacer 4-16 basepairs in length, followed by a secondsequence 16-22 bp in length that is recognized by the TAL-effectordomain (this sequence typically has a 5′ T base). Cleavage by a CompactTALEN produces two base pair 3′ overhangs. In the case of a CRISPR, therecognition sequence is the sequence, typically 16-24 basepairs, towhich the guide RNA binds to direct Cas9 cleavage. Full complementaritybetween the guide sequence and the recognition sequence is notnecessarily required to effect cleavage. Cleavage by a CRISPR canproduce blunt ends (such as by a class II, type II Cas) or overhangingends (such as by a class II, type V Cas), depending on the Cas. In thoseembodiments wherein a Cpf1 Cas is utilized, cleavage by the CRISPRcomplex comprising the same will result in 5′ overhangs and in certainembodiments, 5 nucleotide 5′ overhangs. Each Cas enzyme also requiresthe recognition of a PAM (protospacer adjacent motif) sequence that isnear the recognition sequence complementary to the guide RNA. Theprecise sequence, length requirements for the PAM, and distance from thetarget sequence differ depending on the Cas enzyme, but PAMs aretypically 2-5 base pair sequences adjacent to the target/recognitionsequence. PAM sequences for particular Cas enzymes are known in the art(see, for example, U.S. Pat. No. 8,697,359 and U.S. Publication No.20160208243, each of which is incorporated by reference in its entirety)and PAM sequences for novel or engineered Cas enzymes can be identifiedusing methods known in the art, such as a PAM depletion assay (see, forexample, Karvelis et al. (2017) Methods 121-122:3-8, which isincorporated herein in its entirety). In the case of a zinc finger, theDNA binding domains typically recognize an 18-bp recognition sequencecomprising a pair of nine basepair “half-sites” separated by 2-10basepairs and cleavage by the nuclease creates a blunt end or a 5′overhang of variable length (frequently four basepairs).

As used herein, the term “target site” or “target sequence” refers to aregion of the chromosomal DNA of a cell comprising a recognitionsequence for a nuclease.

As used herein, the term “DNA-binding affinity” or “binding affinity”means the tendency of a meganuclease to non-covalently associate with areference DNA molecule (e.g., a recognition sequence or an arbitrarysequence). Binding affinity is measured by a dissociation constant,K_(d). As used herein, a nuclease has “altered” binding affinity if theK_(d) of the nuclease for a reference recognition sequence is increasedor decreased by a statistically significant percent change relative to areference nuclease.

As used herein, the term “homologous recombination” or “HR” refers tothe natural, cellular process in which a double-stranded DNA-break isrepaired using a homologous DNA sequence as the repair template (see,e.g., Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologousDNA sequence may be an endogenous chromosomal sequence or an exogenousnucleic acid that was delivered to the cell.

As used herein, the term “non-homologous end-joining” or “NHEJ” refersto the natural, cellular process in which a double-stranded DNA-break isrepaired by the direct joining of two non-homologous DNA segments (see,e.g., Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair bynon-homologous end-joining is error-prone and frequently results in theuntemplated addition or deletion of DNA sequences at the site of repair.In some instances, cleavage at a target recognition sequence results inNHEJ at a target recognition site. Nuclease-induced cleavage of a targetsite in the coding sequence of a gene followed by DNA repair by NHEJ canintroduce mutations into the coding sequence, such as frameshiftmutations, that disrupt gene function. Thus, engineered nucleases can beused to effectively knock-out a gene in a population of cells.

As used herein, a “chimeric antigen receptor” or “CAR” refers to anengineered receptor that confers or grafts specificity for an antigenonto an immune effector cell (e.g., a human T cell). A chimeric antigenreceptor typically comprises at least an extracellular ligand-bindingdomain or moiety and an intracellular domain that comprises one or moresignaling domains and/or co-stimulatory domains.

In some embodiments, the extracellular ligand-binding domain or moietyis in the form of a single-chain variable fragment (scFv) derived from amonoclonal antibody, which provides specificity for a particular epitopeor antigen (e.g., an epitope or antigen preferentially present on thesurface of a cell, such as a cancer cell or other disease-causing cellor particle). In some embodiments, the scFv is attached via a linkersequence. In various embodiments, the extracellular ligand-bindingdomain is specific for any antigen or epitope of interest. In someembodiments, the scFv is murine, humanized, or fully human.

The extracellular domain of a chimeric antigen receptor can alsocomprise an autoantigen (see, Payne et al. (2016), Science 353 (6295):179-184), that can be recognized by autoantigen-specific B cellreceptors on B lymphocytes, thus directing T cells to specificallytarget and kill autoreactive B lymphocytes in antibody-mediatedautoimmune diseases. Such CARs can be referred to as chimericautoantibody receptors (CAARs), and their use is encompassed by theinvention.

The extracellular domain of a chimeric antigen receptor can alsocomprise a naturally-occurring ligand for an antigen of interest, or afragment of a naturally-occurring ligand which retains the ability tobind the antigen of interest.

The intracellular stimulatory domain can include one or more cytoplasmicsignaling domains that transmit an activation signal to the immuneeffector cell following antigen binding. Such cytoplasmic signalingdomains can include, without limitation, CD3.

The intracellular stimulatory domain can also include one or moreintracellular co-stimulatory domains that transmit a proliferativeand/or cell-survival signal after ligand binding. As used herein, a“co-stimulatory domain” refers to a polypeptide domain which transmitsan intracellular proliferative and/or cell-survival signal uponactivation. Activation of a co-stimulatory domain may occur followinghomodimerization of two co-stimulatory domain polypeptides. Activationmay also occur, for example, following activation of a constructcomprising the co-stimulatory domain (e.g., a chimeric antigen receptoror an inducible regulatory construct). Generally, a co-stimulatorydomain can be derived from a transmembrane co-stimulatory receptor,particularly from an intracellular portion of a co-stimulatory receptor.Such intracellular co-stimulatory domains can be any of those known inthe art and can include, without limitation, CD27, CD28, CD8, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand thatspecifically binds with CD83, N1, N6, or any combination thereof.

A chimeric antigen receptor can further include additional structuralelements, including a transmembrane domain that is attached to theextracellular ligand-binding domain via a hinge or spacer sequence. Thetransmembrane domain can be derived from any membrane-bound ortransmembrane protein. For example, the transmembrane polypeptide can bea subunit of the T-cell receptor (i.e., an α, β, γ or ζ, polypeptideconstituting CD3 complex), IL2 receptor p55 (a chain), p75 (β chain) orγ chain, subunit chain of Fc receptors (e.g., Fcy receptor III) or CDproteins such as the CD8 alpha chain. Alternatively the transmembranedomain can be synthetic and can comprise predominantly hydrophobicresidues such as leucine and valine.

The hinge region refers to any oligo- or polypeptide that functions tolink the transmembrane domain to the extracellular ligand-bindingdomain. For example, a hinge region may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. Hinge regions may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4 or CD28, or from all or part of an antibody constantregion. Alternatively, the hinge region may be a synthetic sequence thatcorresponds to a naturally occurring hinge sequence, or may be anentirely synthetic hinge sequence. In particular examples, a hingedomain can comprise a part of a human CD8 alpha chain, FcγRIIIa receptoror IgG1.

As used herein, an “exogenous T cell receptor” or “exogenous TCR” refersto a TCR whose sequence is introduced into the genome of an immuneeffector cell (e.g., a human T cell) that may or may not endogenouslyexpress the TCR. Expression of an exogenous TCR on an immune effectorcell can confer specificity for a specific epitope or antigen (e.g., anepitope or antigen preferentially present on the surface of a cancercell or other disease-causing cell or particle). Such exogenous T cellreceptors can comprise alpha and beta chains or, alternatively, maycomprise gamma and delta chains. Exogenous TCRs useful in the inventionmay have specificity to any antigen or epitope of interest.

As used herein, the term “reduced expression” refers to any reduction inthe expression of the endogenous T cell receptor at the cell surface ofa genetically-modified T cell when compared to a control cell. The termreduced can also refer to a reduction in the percentage of cells in apopulation of cells that express an endogenous polypeptide (i.e., anendogenous T cell receptor) at the cell surface when compared to apopulation of control cells. Such a reduction may be up to 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to100%. Accordingly, the term “reduced” encompasses both a partialknockdown and a complete knockdown of the endogenous T cell receptor.

As used herein with respect to both amino acid sequences and nucleicacid sequences, the terms “percent identity,” “sequence identity,”“percentage similarity,” “sequence similarity” and the like refer to ameasure of the degree of similarity of two sequences based upon analignment of the sequences that maximizes similarity between alignedamino acid residues or nucleotides, and that is a function of the numberof identical or similar residues or nucleotides, the number of totalresidues or nucleotides, and the presence and length of gaps in thesequence alignment. A variety of algorithms and computer programs areavailable for determining sequence similarity using standard parameters.As used herein, sequence similarity is measured using the BLASTp programfor amino acid sequences and the BLASTn program for nucleic acidsequences, both of which are available through the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov/), and are described in,for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish andStates (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth.Enzymol. 266:131-141; Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402); Zhang et al. (2000), J. Comput. Biol. 7(1-2):203-14. As usedherein, percent similarity of two amino acid sequences is the scorebased upon the following parameters for the BLASTp algorithm: wordsize=3; gap opening penalty=−11; gap extension penalty=−1; and scoringmatrix=BLOSUM62. As used herein, percent similarity of two nucleic acidsequences is the score based upon the following parameters for theBLASTn algorithm: word size=11; gap opening penalty=−5; gap extensionpenalty=−2; match reward=1; and mismatch penalty=⁻³.

As used herein with respect to modifications of two proteins or aminoacid sequences, the term “corresponding to” is used to indicate that aspecified modification in the first protein is a substitution of thesame amino acid residue as in the modification in the second protein,and that the amino acid position of the modification in the firstproteins corresponds to or aligns with the amino acid position of themodification in the second protein when the two proteins are subjectedto standard sequence alignments (e.g., using the BLASTp program). Thus,the modification of residue “X” to amino acid “A” in the first proteinwill correspond to the modification of residue “Y” to amino acid “A” inthe second protein if residues X and Y correspond to each other in asequence alignment, and despite the fact that X and Y may be differentnumbers.

As used herein, the term “recognition half-site,” “recognition sequencehalf-site,” or simply “half-site” means a nucleic acid sequence in adouble-stranded DNA molecule that is recognized by a monomer of ahomodimeric or heterodimeric meganuclease, or by one subunit of asingle-chain meganuclease.

As used herein, the term “hypervariable region” refers to a localizedsequence within a meganuclease monomer or subunit that comprises aminoacids with relatively high variability. A hypervariable region cancomprise about 50-60 contiguous residues, about 53-57 contiguousresidues, or preferably about 56 residues. In some embodiments, theresidues of a hypervariable region may correspond to positions 24-79 orpositions 215-270 of any one of SEQ ID NOs: 12-27. A hypervariableregion can comprise one or more residues that contact DNA bases in arecognition sequence and can be modified to alter base preference of themonomer or subunit. A hypervariable region can also comprise one or moreresidues that bind to the DNA backbone when the meganuclease associateswith a double-stranded DNA recognition sequence. Such residues can bemodified to alter the binding affinity of the meganuclease for the DNAbackbone and the target recognition sequence. In different embodimentsof the invention, a hypervariable region may comprise between 1-20residues that exhibit variability and can be modified to influence basepreference and/or DNA-binding affinity. In particular embodiments, ahypervariable region comprises between about 15-18 residues that exhibitvariability and can be modified to influence base preference and/orDNA-binding affinity. In some embodiments, variable residues within ahypervariable region correspond to one or more of positions 24, 26, 28,30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ IDNOs: 12-27. In other embodiments, variable residues within ahypervariable region correspond to one or more of positions 215, 217,219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 ofany one of SEQ ID NOs: 12-27.

As used herein, the terms “T cell receptor alpha gene” or “TCR alphagene” are interchangeable and refer to the locus in a T cell whichencodes the T cell receptor alpha subunit. The T cell receptor alpha canrefer to NCBI gene ID number 6955, before or after rearrangement.Following rearrangement, the T cell receptor alpha gene comprises anendogenous promoter, rearranged V and J segments, the endogenous splicedonor site, an intron, the endogenous splice acceptor site, and the TRAClocus, which comprises the subunit coding exons. For example, see FIG.1.

As used herein, the phrases “intron within the T cell receptor alphagene” and “the targeted 5′ intron” refer to the intron, as shown in FIG.1, which in the rearranged T cell receptor alpha gene is positioned 5′upstream of TRAC exon 1, 3′ downstream of the V and J segments, and isflanked by the endogenous splice donor site and the endogenous spliceacceptor site. The targeted 5′ intron can have a sequence comprising SEQID NO: 3, and functional variants thereof which retain the nucleaserecognition sequences encompassed by the invention.

As used herein, the terms “T cell receptor alpha constant region” and“TRAC” are used interchangeably and refer to the coding sequence of theT cell receptor alpha gene. The TRAC includes the wild-type sequence,and functional variants thereof, identified by NCBI Gen ID NO. 28755.

As used herein, the term “endogenous splice donor site” refers to thenaturally-occurring splice donor site positioned 3′ downstream of theendogenous TCR alpha gene promoter and the rearranged V and J segments,and 5′ upstream of the targeted intron. Likewise, the “endogenous spliceacceptor site” refers to the naturally-occurring splice acceptor sitethat is 3′ downstream of the targeted intron and immediately 5′ upstreamof TRAC exon 1. Endogenous splice donor sites and endogenous spliceacceptor sites can be identified in a gene by methods known in the art,such as those described by Desmet et al. (Nucleic Acid Research (2009)37(9): e67). The term “functional” as it relates to the endogenoussplice donor site and the endogenous splice acceptor site refers totheir ability to pair in order to execute splicing of the interveningintron sequence.

As used herein, the term “exogenous splice acceptor site” refers to asplice acceptor site which is comprised by the exogenous sequence ofinterest and is introduced into the targeted 5′ intron. The exogenoussplice acceptor site can comprise a sequence naturally present in thehuman T cell receptor alpha gene, or can comprise a splice acceptorsequence (e.g., a consensus or heterologous sequence) which is notnaturally present in the gene. The exogenous splice acceptor site mayfurther comprise an exogenous branch site if necessary to promotesplicing of the intron. Such a branch site may comprise a sequence whichis naturally present in the T cell receptor alpha gene, or can comprisea branch site sequence (e.g., a consensus or heterologous sequence)which is not naturally present in the gene.

The terms “recombinant DNA construct,” “recombinant construct,”“expression cassette,” “expression construct,” “chimeric construct,”“construct,” and “recombinant DNA fragment” are used interchangeablyherein and are single or double-stranded polynucleotides. A recombinantconstruct comprises an artificial combination of single ordouble-stranded polynucleotides, including, without limitation,regulatory and coding sequences that are not found together in nature.For example, a recombinant DNA construct may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource and arranged in a manner different than that found in nature.Such a construct may be used by itself or may be used in conjunctionwith a vector.

As used herein, a “vector” or “recombinant DNA vector” may be aconstruct that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. If a vector is used then the choice ofvector is dependent upon the method that will be used to transform hostcells as is well known to those skilled in the art. Vectors can include,without limitation, plasmid vectors and recombinant AAV vectors, or anyother vector known in that art suitable for delivering a gene encoding ameganuclease of the invention to a target cell. The skilled artisan iswell aware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleotides or nucleic acid sequences ofthe invention.

As used herein, a “vector” can also refer to a viral vector. Viralvectors can include, without limitation, retroviral vectors, lentiviralvectors, adenoviral vectors, and adeno-associated viral vectors (AAV).

As used herein, a “polycistronic” mRNA refers to a single messenger RNAthat comprises two or more coding sequences (i.e., cistrons) and encodesmore than one protein. A polycistronic mRNA can comprise any elementknown in the art to allow for the translation of two or more genes fromthe same mRNA molecule including, but not limited to, an IRES element, aT2A element, a P2A element, an E2A element, and an F2A element.

As used herein, a “human T cell” or “T cell” refers to a T cell isolatedfrom a donor, particularly a human donor. T cells, and cells derivedtherefrom, include isolated T cells that have not been passaged inculture, T cells that have been passaged and maintained under cellculture conditions without immortalization, and T cells that have beenimmortalized and can be maintained under cell culture conditionsindefinitely.

As used herein, a “human natural killer cell” or “human NK cell” or“natural killer cell” or “NK cell” refers to a type of cytotoxiclymphocyte critical to the innate immune system. The role NK cells playis analogous to that of cytotoxic T-cells in the vertebrate adaptiveimmune response. NK cells provide rapid responses to virally infectedcells and respond to tumor formation, acting at around 3 days afterinfection.

As used herein, a “control” or “control cell” refers to a cell thatprovides a reference point for measuring changes in genotype orphenotype of a genetically-modified cell. A control cell may comprise,for example: (a) a wild-type cell, i.e., of the same genotype as thestarting material for the genetic alteration that resulted in thegenetically-modified cell; (b) a cell of the same genotype as thegenetically-modified cell but that has been transformed with a nullconstruct (i.e., with a construct that has no known effect on the traitof interest); or, (c) a cell genetically identical to thegenetically-modified cell but that is not exposed to conditions orstimuli or further genetic modifications that would induce expression ofaltered genotype or phenotype.

As used herein, the terms “treatment” or “treating a subject” refers tothe administration of a genetically-modified T cell of the invention toa subject having a disease. For example, the subject can have a diseasesuch as cancer, and treatment can represent immunotherapy for thetreatment of the disease. Desirable effects of treatment include, butare not limited to, preventing occurrence or recurrence of disease,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some aspects, a genetically-modifiedcell described herein is administered during treatment in the form of apharmaceutical composition of the invention.

The term “effective amount” or “therapeutically effective amount” refersto an amount sufficient to effect beneficial or desirable biologicaland/or clinical results. The therapeutically effective amount will varydepending on the formulation or composition used, the disease and itsseverity and the age, weight, physical condition and responsiveness ofthe subject to be treated.

As used herein, the term “cancer” should be understood to encompass anyneoplastic disease (whether invasive or metastatic) which ischaracterized by abnormal and uncontrolled cell division causingmalignant growth or tumor.

As used herein, the term “carcinoma” refers to a malignant growth madeup of epithelial cells.

As used herein, the term “leukemia” refers to malignancies of thehematopoietic organs/systems and is generally characterized by anabnormal proliferation and development of leukocytes and theirprecursors in the blood and bone marrow.

As used herein, the term “sarcoma” refers to a tumor which is made up ofa substance like the embryonic connective tissue and is generallycomposed of closely packed cells embedded in a fibrillary,heterogeneous, or homogeneous substance.

As used herein, the term “melanoma” refers to a tumor arising from themelanocytic system of the skin and other organs.

As used herein, the term “lymphoma” refers to a group of blood celltumors that develop from lymphocytes.

As used herein, the term “blastoma” refers to a type of cancer that iscaused by malignancies in precursor cells or blasts (immature orembryonic tissue).

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable thatis inherently discrete, the variable can be equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable that is inherently continuous, the variablecan be equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable that is described as having values between 0 and 2 can take thevalues 0, 1, or 2 if the variable is inherently discrete, and can takethe values 0.0, 0.1, 0.01, 0.001, or any other real values ≥0 and ≤2 ifthe variable is inherently continuous.

2.1 Principle of the Invention

The present invention is based, in part, on the discovery that insertionof a sequence of interest (including an exogenous splice acceptor siteand/or a poly A signal) into a nuclease cleavage site in the targeted 5′intron of the T cell receptor alpha gene allows for the production ofTCR− cells only when the insert is present. If no insert is present, thenuclease-modified intron is simply removed and the endogenous gene isexpressed. Thus, in an example where the sequence of interest includes aCAR coding sequence, the present invention provides a method forproducing a population where most or all TCR− cells are TCR−/CAR+ cells.Any other peptide of interest can be expressed from the sequence ofinterest in the same manner as a CAR.

By contrast, conventional nuclease-based approaches for generatingmodified T cells target coding sequences of the TRAC and/or theendogenous splice acceptor site 5′ upstream of TRAC exon 1.Consequently, they can generate highly mixed populations of TCR− cells,which include a substantial percentage of TCR−/CAR− cells, due to NHEJat the nuclease cleavage site which creates indels and disrupts proteinexpression.

Thus, by reducing the need to purify a mixed population of TCR− cells,the present invention provides a simplified method for producing apopulation of allogeneic CAR T cells that express an antigen-specificCAR and have reduced expression of the endogenous TCR. Such cells canexhibit reduced or no induction of graft-versus-host-disease (GVHD) whenadministered to an allogeneic subject. Furthermore, the inclusion of a2A element in the exogenous sequence of interest allows for expressionof a coding sequence to be driven by the endogenous T cell receptoralpha gene promoter, rather than by an exogenous promoter. In thismanner, expression of a polypeptide such as a CAR can be regulated bythe T cell feedback mechanisms normally associated with TCR expression.

2.2 Nucleases for Recognizing and Cleaving Recognition Sequences withinthe Targeted 5′ Intron of the T Cell Receptor Alpha Gene

It is known in the art that it is possible to use a site-specificnuclease to make a DNA break in the genome of a living cell, and thatsuch a DNA break can result in permanent modification of the genome viamutagenic NHEJ repair or via homologous recombination with a transgenicDNA sequence. NHEJ can produce mutagenesis at the cleavage site,resulting in inactivation of the allele. NHEJ-associated mutagenesis mayinactivate an allele via generation of early stop codons, frameshiftmutations producing aberrant non-functional proteins, or could triggermechanisms such as nonsense-mediated mRNA decay. The use of nucleases toinduce mutagenesis via NHEJ can be used to target a specific mutation ora sequence present in a wild-type allele. The use of nucleases to inducea double-strand break in a target locus is known to stimulate homologousrecombination, particularly of transgenic DNA sequences flanked bysequences that are homologous to the genomic target. In this manner,exogenous nucleic acid sequences can be inserted into a target locus.Such exogenous nucleic acids can encode, for example, a chimeric antigenreceptor, an exogenous TCR, or any sequence or polypeptide of interest.

In different embodiments, a variety of different types of nucleases areuseful for practicing the invention. In one embodiment, the inventioncan be practiced using engineered meganucleases. In another embodiment,the invention can be practiced using a CRISPR nuclease or CRISPRNickase. Methods for making CRISPRs and CRISPR Nickases that recognizepre-determined DNA sites are known in the art, for example Ran, et al.(2013) Nat Protoc. 8:2281-308. In another embodiment, the invention canbe practiced using TALENs or Compact TALENs. Methods for making TALEdomains that bind to pre-determined DNA sites are known in the art, forexample Reyon et al. (2012) Nat Biotechnol. 30:460-5. In anotherembodiment, the invention can be practiced using zinc finger nucleases(ZFNs). In a further embodiment, the invention can be practiced usingmegaTALs.

In preferred embodiments, the nucleases used to practice the inventionare single-chain meganucleases. A single-chain meganuclease comprises anN-terminal subunit and a C-terminal subunit joined by a linker peptide.Each of the two domains recognizes half of the recognition sequence(i.e., a recognition half-site) and the site of DNA cleavage is at themiddle of the recognition sequence near the interface of the twosubunits. DNA strand breaks are offset by four base pairs such that DNAcleavage by a meganuclease generates a pair of four base pair, 3′single-strand overhangs.

In some examples, engineered meganucleases of the invention have beenengineered to recognize and cleave the TRC 11-12 recognition sequence(SEQ ID NO: 4). Such engineered meganucleases are collectively referredto herein as “TRC 11-12 meganucleases.” Exemplary TRC 11-12meganucleases are provided in SEQ ID NOs: 12-15.

In additional examples, engineered meganucleases of the invention havebeen engineered to recognize and cleave the TRC 15-16 recognitionsequence (SEQ ID NO: 6). Such engineered meganucleases are collectivelyreferred to herein as “TRC 15-16 meganucleases.” Exemplary TRC 15-16meganucleases are provided in SEQ ID NOs: 16-19.

In additional examples, engineered meganucleases of the invention havebeen engineered to recognize and cleave the TRC 17-18 recognitionsequence (SEQ ID NO: 8). Such engineered meganucleases are collectivelyreferred to herein as “TRC 17-18 meganucleases.” Exemplary TRC 17-18meganucleases are provided in SEQ ID NOs: 20-23.

In further examples, engineered meganucleases of the invention have beenengineered to recognize and cleave the TRC 19-20 recognition sequence(SEQ ID NO: 10). Such engineered meganucleases are collectively referredto herein as “TRC 19-20 meganucleases.” Exemplary TRC 19-20meganucleases are provided in SEQ ID NOs: 24-27.

Engineered meganucleases of the invention comprise a first subunit,comprising a first hypervariable (HVR1) region, and a second subunit,comprising a second hypervariable (HVR2) region. Further, the firstsubunit binds to a first recognition half-site in the recognitionsequence (e.g., the TRC11, TRC15, TRC17, or TRC19 half-site), and thesecond subunit binds to a second recognition half-site in therecognition sequence (e.g., the TRC12, TRC16, TRC18, or TRC20half-site). In embodiments where the recombinant meganuclease is asingle-chain meganuclease, the first and second subunits can be orientedsuch that the first subunit, which comprises the HVR1 region and bindsthe first half-site, is positioned as the N-terminal subunit, and thesecond subunit, which comprises the HVR2 region and binds the secondhalf-site, is positioned as the C-terminal subunit. In alternativeembodiments, the first and second subunits can be oriented such that thefirst subunit, which comprises the HVR1 region and binds the firsthalf-site, is positioned as the C-terminal subunit, and the secondsubunit, which comprises the HVR2 region and binds the second half-site,is positioned as the N-terminal subunit. Exemplary TRC 11-12meganucleases of the invention are provided in Table 1. Exemplary TRC15-16 meganucleases of the invention are provided in Table 2. ExemplaryTRC 17-18 meganucleases of the invention are provided in Table 3.Exemplary TRC 19-20 meganucleases of the invention are provided in Table4.

TABLE 1 Exemplary engineered meganucleases engineered to recognize andcleave the TRC 1-2 recognition sequence (SEQ ID NO:4) AA TRC11 TRC11*TRC11 TRC12 TRC12 *TRC12 SEQ Subunit Subunit Subunit Subunit SubunitSubunit Meganuclease ID Residues SEQ ID % Residues SEQ ID % TRC 11-12 ×.4  12 198-344 28 100 7-153 32 100 TRC 11-12 × .82 13 198-344 29 92.527-153 33 93.2 TRC 11-12 × .60 14 198-344 30 89.8 7-153 34 99.32 TRC11-12 × .63 15 198-344 31 91.84 7-153 35 95.24 *“TRC11 Subunit %” and“TRC12 Subunit %” represent the amino acid sequence identity between theTRC11-binding and TRC12-binding subunit regions of each meganuclease andthe TRC11-binding and TRC12-binding subunit regions, respectively, ofthe TRC 11-12 × .4 meganuclease.

TABLE 2 Exemplary engineered meganucleases engineered to recognize andcleave the TRC 15-16 recognition sequence (SEQ ID NO:6) AA TRC15 TRC15*TRC15 TRC16 TRC16 *TRC16 SEQ Subunit Subunit Subunit Subunit SubunitSubunit Meganuclease ID Residues SEQ ID % Residues SEQ ID % TRC 15-16 ×.31 16 7-153 36 100 198-344 40 100 TRC 15-16 × .87 17 7-153 37 100198-344 41 92.52 TRC 15-16 × .63 18 7-153 38 98.64 198-344 42 92.52 TRC15-16 × .89 19 7-153 39 99.32 198-344 43 99.32 *“TRC15 Subunit %” and“TRC16 Subunit %” represent the amino acid sequence identity between theTRC15-binding and TRC16-binding subunit regions of each meganuclease andthe TRC15-binding and TRC16-binding subunit regions, respectively, ofthe TRC 15-16 × .31 meganuclease.

TABLE 3 Exemplary engineered meganucleases engineered to recognize andcleave the TRC 17-18 recognition sequence (SEQ ID NO:8) AA TRC17 TRC17*TRC17 TRC18 TRC18 *TRC18 SEQ Subunit Subunit Subunit Subunit SubunitSubunit Meganuclease ID Residues SEQ ID % Residues SEQ ID % TRC 17-18 ×.15 20 7-153 44 100 198-344 48 100 TRC 17-18 × .82 21 7-153 45 90.48198-344 49 89.8 TRC 17-18 × .18 22 7-153 46 91.16 198-344 50 95.24 TRC17-18 × .71 23 7-153 47 91.16 198-344 51 94.56 *“TRC17 Subunit %” and“TRC18 Subunit %” represent the amino acid sequence identity between theTRC17-binding and TRC18-binding subunit regions of each meganuclease andthe TRC17-binding and TRC18-binding subunit regions, respectively, ofthe TRC17-18 × .15 meganuclease.

TABLE 4 Exemplary engineered meganucleases engineered to recognize andcleave the TRC 19-20 recognition sequence (SEQ ID NO:10) AA TRC19 TRC19*TRC19 TRC20 TRC20 *TRC20 SEQ Subunit Subunit Subunit Subunit SubunitSubunit Meganuclease ID Residues SEQ ID % Residues SEQ ID % TRC 19-20 ×.85 24 7-153 52 100 198-344 56 100 TRC 19-20 × .74 25 7-153 53 94.56198-344 57 94.56 TRC 19-20 × .71 26 7-153 54 93.88 198-344 58 89.8 TRC19-20 × .87 27 7-153 55 100 198-344 59 92.52 *“TRC19 Subunit %” and“TRC20 Subunit %” represent the amino acid sequence identity between theTRC19-binding and TRC20-binding subunit regions of each meganuclease andthe TRC19-binding and TRC20-binding subunit regions, respectively, ofthe TRC 19-20 × .85 meganuclease.

2.3 Methods for Producing Genetically-Modified Cells

Following rearrangement, the human T cell receptor alpha gene comprisesa number of elements. Generally, without being bound by any specifictheory, these elements include from 5′ to 3′, an endogenous promoter,rearranged V and J segments, an endogenous splice donor site, an intron(i.e., the targeted 5′ intron), an endogenous splice acceptor site, andthe TRAC locus, which comprises the alpha subunit coding exons andinterspaced introns. For example, see FIG. 1.

The invention disclosed herein provides methods for producinggenetically-modified T cells comprising a modified TCR alpha gene. Tcells can be obtained from a number of sources, including peripheralblood mononuclear cells, bone marrow, lymph node tissue, cord blood,thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In certain embodiments of thepresent disclosure, any number of T cell lines available in the art maybe used. In some embodiments of the present disclosure, T cells areobtained from a unit of blood collected from a subject using any numberof techniques known to the skilled artisan. In one embodiment, cellsfrom the circulating blood of an individual are obtained by apheresis.

The modified T cell receptor alpha gene comprises an exogenous sequenceof interest inserted into the intron within the TCR alpha gene that ispositioned 5′ upstream of TRAC exon 1 (i.e., the targeted 5′ intron).More specifically, the exogenous sequence of interest can be inserted 3′downstream of the rearranged V and J segments and the endogenous splicedonor site, and 5′ upstream of the endogenous splice acceptor site. Inspecific embodiments, the targeted 5′ intron comprises the sequence setforth in SEQ ID NO: 3, or a sequence having at least 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acidsequence set forth in SEQ ID NO: 3 and comprising a recognition sequenceof an engineered nuclease as described herein.

In some embodiments, the exogenous sequence of interest can be insertedinto the intron at a double-stranded cleavage site generated by anengineered nuclease, such as an engineered meganuclease, a zinc fingernuclease, a TALEN, a compact TALEN, a CRISPR nuclease, or a megaTAL.Cleavage sites generated by such nucleases can allow for homologousrecombination of the exogenous sequence of interest directly into the 5′intron.

The “endogenous splice donor site” refers to the naturally-occurringsplice donor site that is 3′ downstream of the endogenous TCR alpha genepromoter and the rearranged V and J segments, and 5′ upstream of thetargeted intron. Likewise, the “endogenous splice acceptor site” refersto the naturally-occurring splice acceptor site that is 3′ downstream ofthe targeted intron and immediately 5′ upstream of TRAC exon 1. See,FIG. 1.

In specific embodiments, the engineered nucleases disclosed herein donot modify either the endogenous splice donor site or the endogenoussplice acceptor site, as both sites should retain their functionality topractice the invention. In some embodiments, the endogenous splice donorsite and/or the endogenous splice acceptor site can be modified as longas each site retains the ability to pair with the other and splice theintron (i.e., retains functionality). Thus, as used herein, a functionalendogenous splice donor site has the ability to pair with the endogenoussplice acceptor site to remove the intron. Likewise, as used herein, afunctional endogenous splice acceptor site has the ability to pair withthe endogenous splice donor site to remove the intron.

In particular embodiments, the sequence of interest can comprise anexogenous splice acceptor site. As used herein, the term “exogenous” or“heterologous” in reference to a nucleotide sequence is intended to meana sequence that is purely synthetic, that originates from a foreignspecies, or, if from the same species, is substantially modified fromits native form in composition and/or genomic locus by deliberate humanintervention. Thus, the exogenous splice acceptor site can be a purelysynthetic splice acceptor site, or a splice acceptor site from the humangenome that has been modified in sequence or genomic locus.

In specific embodiments, the exogenous splice acceptor site is able topartner with the endogenous splice donor site in order to splice out theintervening intron sequence. In this manner, the exogenous spliceacceptor site can disrupt natural splicing of the targeted 5′ intron bycompeting with the endogenous splice acceptor site for partnering withthe endogenous splice donor site.

In various embodiments, the exogenous sequence of interest can comprisea coding sequence for a protein of interest. It is envisioned that thecoding sequence can be for any protein of interest.

In certain embodiments, the exogenous sequence of interest comprises anucleic acid sequence encoding a CAR. Generally, a CAR of the presentdisclosure will comprise at least an extracellular domain and anintracellular domain. In some embodiments, the extracellular domaincomprises a target-specific binding element otherwise referred to as aligand-binding domain or moiety. In some embodiments, the intracellulardomain, or cytoplasmic domain, comprises at least one co-stimulatorydomain and one or more signaling domains such as, for example, CD3. Inother embodiments, the CAR may only comprise a signaling domain, such asCD3, and the cell may comprise one or more co-stimulatory domains onanother construct expressed in the cell.

In some embodiments, a CAR useful in the invention comprises anextracellular, target-specific binding element otherwise referred to asa ligand-binding domain or moiety. The choice of ligand-binding domaindepends upon the type and number of ligands that define the surface of atarget cell. For example, the ligand-binding domain may be chosen torecognize a ligand that acts as a cell surface marker on target cellsassociated with a particular disease state. Thus, examples of cellsurface markers that may act as ligands for the ligand-binding domain ina CAR can include those associated with viruses, bacterial and parasiticinfections, autoimmune disease, and cancer cells. In some embodiments, aCAR is engineered to target a tumor-specific antigen of interest by wayof engineering a desired ligand-binding moiety that specifically bindsto an antigen on a tumor cell. In the context of the present disclosure,“tumor antigen” refers to antigens that are common to specifichyperproliferative disorders such as cancer.

In some embodiments, the extracellular ligand-binding domain of the CARis specific for any antigen or epitope of interest, particularly anytumor antigen or epitope of interest. As non-limiting examples, in someembodiments the antigen of the target is a tumor-associated surfaceantigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA),epithelial cell adhesion molecule (EpCAM), epidermal growth factorreceptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD22, CD30,CD40, CLL1, disialoganglioside GD2, ductal-epithelial mucine, gp36,TAG-72, glycosphingolipids, glioma-associated antigen, B-human chorionicgonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53,prostein, PSMA, surviving and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulingrowth factor (IGF1)-1, IGF-II, IGFI receptor, mesothelin, a majorhistocompatibility complex (MHC) molecule presenting a tumor-specificpeptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, theextra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1domain of tenascin-C (TnC A1) and fibroblast associated protein (fap); alineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24,CD25, CD33, CD34, CD38, CD123, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2(CD86), endoglin, a major histocompatibility complex (MHC) molecule,BCMA (CD269, TNFRSF 17), CS1, or a virus-specific surface antigen suchas an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen,a CMV-specific antigen, a HPV-specific antigen such as the E6 or E7oncoproteins, a Lasse Virus-specific antigen, an InfluenzaVirus-specific antigen, as well as any derivate or variant of thesesurface markers. In a particular embodiment of the present disclosure,the ligand-binding domain is specific for CD19.

In some embodiments, the extracellular domain of a chimeric antigenreceptor further comprises an autoantigen (see, Payne et al. (2016)Science, Vol. 353 (6295): 179-184), which can be recognized byautoantigen-specific B cell receptors on B lymphocytes, thus directing Tcells to specifically target and kill autoreactive B lymphocytes inantibody-mediated autoimmune diseases. Such CARs can be referred to aschimeric autoantibody receptors (CAARs).

In some embodiments, the extracellular domain of a chimeric antigenreceptor can comprise a naturally-occurring ligand for an antigen ofinterest, or a fragment of a naturally-occurring ligand which retainsthe ability to bind the antigen of interest.

In some embodiments, a CAR comprises a transmembrane domain which linksthe extracellular ligand-binding domain or autoantigen with theintracellular signaling and co-stimulatory domains via a hinge or spacersequence. The transmembrane domain can be derived from anymembrane-bound or transmembrane protein. For example, the transmembranepolypeptide can be a subunit of the T-cell receptor (i.e., an α, β, γ orζ, polypeptide constituting CD3 complex), IL2 receptor p55 (a chain),p75 (β chain) or γ chain, subunit chain of Fc receptors (e.g., Fcyreceptor III) or CD proteins such as the CD8 alpha chain. Alternativelythe transmembrane domain can be synthetic and can comprise predominantlyhydrophobic residues such as leucine and valine. In particular examples,the transmembrane domain is a CD8a transmembrane polypeptide.

The hinge region refers to any oligo- or polypeptide that functions tolink the transmembrane domain to the extracellular ligand-bindingdomain. For example, a hinge region may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. Hinge regions may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4 or CD28, or from all or part of an antibody constantregion. Alternatively, the hinge region may be a synthetic sequence thatcorresponds to a naturally occurring hinge sequence, or may be anentirely synthetic hinge sequence. In particular examples, a hingedomain can comprise a part of a human CD8 alpha chain, FcγRIIIa receptoror IgG1.

Intracellular signaling domains of a CAR of are responsible foractivation of at least one of the normal effector functions of the cellin which the CAR has been placed and/or activation of proliferative andcell survival pathways. The term “effector function” refers to aspecialized function of a cell. Effector function of a T cell, forexample, may be cytolytic activity or helper activity including thesecretion of cytokines. An intracellular signaling domain, such as CD3,can provide an activation signal to the cell in response to binding ofthe extracellular domain. As discussed, the activation signal can inducean effector function of the cell such as, for example, cytolyticactivity or cytokine secretion.

The intracellular domain of the CAR can include one or moreintracellular co-stimulatory domains which transmit a co-stimulatorysignal to promote cell proliferation, cell survival, and/or cytokinesecretion after binding of the extracellular domain. Such intracellularco-stimulatory domains include those known in the art such as, withoutlimitation, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3 and a ligand that specifically binds with CD83, N1, or N6.

The CAR can be specific for any type of cancer cell. Such cancers caninclude, without limitation, carcinoma, lymphoma, sarcoma, blastomas,leukemia, cancers of B cell origin, breast cancer, gastric cancer,neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer,colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma,leukemia, and Hodgkin's lymphoma. In certain embodiments, cancers of Bcell origin include, without limitation, B lineage acute lymphoblasticleukemia, B cell chronic lymphocytic leukemia, B cell non-Hodgkin'slymphoma, and multiple myeloma.

The sequence of interest can further encode an exogenous T cell receptor(TCR). Such exogenous T cell receptors can comprise alpha and betachains or, alternatively, may comprise gamma and delta chains. ExogenousTCRs useful in the invention may have specificity to any antigen orepitope of interest.

In other embodiments, the sequence of interest can encode the wild-typeor modified version of an endogenous gene of interest.

The sequence of interest can comprise an element or peptide known in theart to allow for the translation of two more genes from the same mRNAmolecule, including but not limited to IRES elements and 2A elements,such as, a T2A element, a P2A element, an E2A element, and an F2Aelement. In specific embodiments, such elements in the exogenoussequence of interest can be located 5′ upstream of a nucleic acidsequence encoding a protein of interest (e.g. a CAR).

The exogenous sequence of interest described herein can further compriseadditional control sequences. For example, the sequences of interest caninclude homologous recombination enhancer sequences, Kozak sequences,polyadenylation sequences, transcriptional termination sequences,selectable marker sequences (e.g., antibiotic resistance genes), originsof replication, and the like. Sequences of interest described herein canalso include at least one nuclear localization signal. Examples ofnuclear localization signals are known in the art (see, e.g., Lange etal., J. Biol. Chem., 2007, 282:5101-5105).

In specific embodiments, the exogenous sequence of interest comprises apolyadenylation sequence or poly A signal. Thus, a sequence of interestcan comprise a poly A signal located 3′ downstream of a sequenceencoding a protein of interest (e.g. a CAR). In this manner,transcription of the T cell receptor alpha gene, particularly the codingsequences of the TRAC locus, will be disrupted by the poly A signal,thus preventing expression of the T cell receptor alpha subunit.

In some examples of the invention, the exogenous sequence of interestincludes, from 5′ to 3′, an exogenous splice acceptor site, a 2A elementor IRES element, a coding sequence for a protein of interest, and apolyA signal. In certain examples, the exogenous sequence of interestincludes, from 5′ to 3′, an exogenous splice acceptor site, a 2A elementor IRES element, a coding sequence for a CAR or an exogenous T cellreceptor, and a polyA signal. In some examples, the exogenous sequenceof interest can further include an exogenous branch site positioned 5′upstream of the exogenous splice acceptor site. In the various examplesof the invention, the 2A element can be, without limitation, a T2A, aP2A, an E2A, or an F2A element.

Engineered nucleases of the invention can be delivered into a cell inthe form of protein or, preferably, as a nucleic acid encoding theengineered nuclease. Such nucleic acid can be DNA (e.g., circular orlinearized plasmid DNA or PCR products) or RNA (e.g., mRNA). Forembodiments in which the engineered nuclease coding sequence isdelivered in DNA form, it should be operably linked to a promoter tofacilitate transcription of the nuclease gene. Mammalian promoterssuitable for the invention include constitutive promoters such as thecytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc NatlAcad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist andChambon (1981), Nature. 290(5804):304-10) as well as inducible promoterssuch as the tetracycline-inducible promoter (Dingermann et al. (1992),Mol Cell Biol. 12(9):4038-45).

In some embodiments, mRNA encoding the engineered nuclease is deliveredto the cell because this reduces the likelihood that the gene encodingthe engineered nuclease will integrate into the genome of the cell. SuchmRNA encoding an engineered nuclease can be produced using methods knownin the art such as in vitro transcription. In some embodiments, the mRNAis capped using 7-methyl-guanosine. In some embodiments, the mRNA may bepolyadenylated.

In particular embodiments, an mRNA encoding an engineered nuclease ofthe invention can be a polycistronic mRNA encoding two or more nucleasesthat are simultaneously expressed in the cell. A polycistronic mRNA canencode two or more nucleases of the invention that target differentrecognition sequences in the same target gene. Alternatively, apolycistronic mRNA can encode at least one nuclease described herein andat least one additional nuclease targeting a separate recognitionsequence positioned in the same gene, or targeting a second recognitionsequence positioned in a second gene such that cleavage sites areproduced in both genes. A polycistronic mRNA can comprise any elementknown in the art to allow for the translation of two or more genes(i.e., cistrons) from the same mRNA molecule including, but not limitedto, an IRES element, a T2A element, a P2A element, an E2A element, andan F2A element.

Purified nuclease proteins can be delivered into cells to cleave genomicDNA, which allows for homologous recombination or non-homologousend-joining at the cleavage site with a sequence of interest, by avariety of different mechanisms known in the art.

In some embodiments, engineered nuclease proteins, or DNA/mRNA encodingengineered nucleases, are coupled to a cell penetrating peptide ortargeting ligand to facilitate cellular uptake. Examples of cellpenetrating peptides known in the art include poly-arginine(Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), TAT peptidefrom the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 25: 679-736),MPG (Simeoni, et al. (2003) Nucleic Acids Res. 31:2717-2724), Pep-1(Deshayes et al. (2004) Biochemistry 43: 7698-7706, and HSV-1 VP-22(Deshayes et al. (2005) Cell Mol Life Sci. 62:1839-49. In an alternativeembodiment, engineered nucleases, or DNA/mRNA encoding engineerednucleases, are coupled covalently or non-covalently to an antibody thatrecognizes a specific cell surface receptor expressed on target cellssuch that the nuclease protein/DNA/mRNA binds to and is internalized bythe target cells. Alternatively, engineered nuclease protein/DNA/mRNAcan be coupled covalently or non-covalently to the natural ligand (or aportion of the natural ligand) for such a cell surface receptor.(McCall, et al. (2014) Tissue Barriers. 2(4):e944449; Dinda, et al.(2013) Curr Pharm Biotechnol. 14:1264-74; Kang, et al. (2014) Curr PharmBiotechnol. 15(3):220-30; Qian et al. (2014) Expert Opin Drug MetabToxicol. 10(11):1491-508).

In some embodiments, engineered nuclease proteins, or DNA/mRNA encodingengineered nucleases, are coupled covalently or, preferably,non-covalently to a nanoparticle or encapsulated within such ananoparticle using methods known in the art (Sharma, et al. (2014)Biomed Res Int. 2014). A nanoparticle is a nanoscale delivery systemwhose length scale is <1 μm, preferably <100 nm. Such nanoparticles maybe designed using a core composed of metal, lipid, polymer, orbiological macromolecule, and multiple copies of the recombinantmeganuclease proteins, mRNA, or DNA can be attached to or encapsulatedwith the nanoparticle core. This increases the copy number of theprotein/mRNA/DNA that is delivered to each cell and, so, increases theintracellular expression of each engineered nuclease to maximize thelikelihood that the target recognition sequences will be cut. Thesurface of such nanoparticles may be further modified with polymers orlipids (e.g., chitosan, cationic polymers, or cationic lipids) to form acore-shell nanoparticle whose surface confers additional functionalitiesto enhance cellular delivery and uptake of the payload (Jian et al.(2012) Biomaterials. 33(30): 7621-30). Nanoparticles may additionally beadvantageously coupled to targeting molecules to direct the nanoparticleto the appropriate cell type and/or increase the likelihood of cellularuptake. Examples of such targeting molecules include antibodies specificfor cell surface receptors and the natural ligands (or portions of thenatural ligands) for cell surface receptors.

In some embodiments, the engineered nucleases or DNA/mRNA encoding theengineered nucleases, are encapsulated within liposomes or complexedusing cationic lipids (see, e.g., Lipofectamine, Life TechnologiesCorp., Carlsbad, Calif.; Zuris et al. (2015) Nat Biotechnol. 33: 73-80;Mishra et al. (2011) J Drug Deliv. 2011:863734). The liposome andlipoplex formulations can protect the payload from degradation, andfacilitate cellular uptake and delivery efficiency through fusion withand/or disruption of the cellular membranes of the cells.

In some embodiments, engineered nuclease proteins, or DNA/mRNA encodingengineered nucleases, are encapsulated within polymeric scaffolds (e.g.,PLGA) or complexed using cationic polymers (e.g., PEI, PLL) (Tamboli etal. (2011) Ther Deliv. 2(4): 523-536).

In some embodiments, engineered nuclease proteins, or DNA/mRNA encodingengineered nucleases, are combined with amphiphilic molecules thatself-assemble into micelles (Tong et al. (2007) J Gene Med. 9(11):956-66). Polymeric micelles may include a micellar shell formed with ahydrophilic polymer (e.g., polyethyleneglycol) that can preventaggregation, mask charge interactions, and reduce nonspecificinteractions outside of the cell.

In some embodiments, engineered nuclease proteins, or DNA/mRNA encodingengineered nucleases, are formulated into an emulsion or a nanoemulsion(i.e., having an average particle diameter of <1 nm) for delivery to thecell. The term “emulsion” refers to, without limitation, anyoil-in-water, water-in-oil, water-in-oil-in-water, oroil-in-water-in-oil dispersions or droplets, including lipid structuresthat can form as a result of hydrophobic forces that drive apolarresidues (e.g., long hydrocarbon chains) away from water and polar headgroups toward water, when a water immiscible phase is mixed with anaqueous phase. These other lipid structures include, but are not limitedto, unilamellar, paucilamellar, and multilamellar lipid vesicles,micelles, and lamellar phases. Emulsions are composed of an aqueousphase and a lipophilic phase (typically containing an oil and an organicsolvent). Emulsions also frequently contain one or more surfactants.Nanoemulsion formulations are well known, e.g., as described in USPatent Application Nos. 2002/0045667 and 2004/0043041, and U.S. Pat.Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, each of which isincorporated herein by reference in its entirety.

In some embodiments, engineered nuclease proteins, or DNA/mRNA encodingengineered nucleases, are covalently attached to, or non-covalentlyassociated with, multifunctional polymer conjugates, DNA dendrimers, andpolymeric dendrimers (Mastorakos et al. (2015) Nanoscale. 7(9): 3845-56;Cheng et al. (2008) J Pharm Sci. 97(1): 123-43). The dendrimergeneration can control the payload capacity and size, and can provide ahigh payload capacity. Moreover, display of multiple surface groups canbe leveraged to improve stability and reduce nonspecific interactions.

In some embodiments, genes encoding an engineered nuclease and/orsequences of interest are introduced into a cell using a viral vector.Such vectors are known in the art and include retroviral vectors,lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV)vectors (reviewed in Vannucci, et al. (2013 New Microbiol. 36:1-22).Recombinant AAV vectors useful in the invention can have any serotypethat allows for transduction of the virus into the cell and insertion ofthe nuclease gene into the cell genome. In particular embodiments,recombinant AAV vectors have a serotype of AAV2 or AAV6. Recombinant AAVvectors can also be self-complementary such that they do not requiresecond-strand DNA synthesis in the host cell (McCarty, et al. (2001)Gene Ther. 8:1248-54).

If the engineered nuclease genes are delivered in DNA form (e.g.plasmid) and/or via a viral vector (e.g. AAV) they must be operablylinked to a promoter. In some embodiments, this can be a viral promotersuch as endogenous promoters from the viral vector (e.g. the LTR of alentiviral vector) or the well-known cytomegalovirus- or SV40virus-early promoters. In a preferred embodiment, nuclease genes areoperably linked to a promoter that drives gene expression preferentiallyin the target cell (e.g., a T cell).

The invention further provides for the introduction of an exogenoussequence of interest into the T cell receptor alpha gene, particularlyinto a recognition sequence within the targeted 5′ intron. In someembodiments, the exogenous sequence of interest comprises a 5′ homologyarm and a 3′ homology arm flanking the elements of the insert (i.e., theexogenous splice acceptor site, the IRES or 2A element, the codingsequence for a protein of interest, and/or the poly A signal). Suchhomology arms have sequence homology to corresponding sequences 5′upstream and 3′ downstream of the nuclease recognition sequence in thetargeted 5′ intron where a cleavage site is produced. In general,homology arms can have a length of at least 50 base pairs, preferably atleast 100 base pairs, and up to 2000 base pairs or more, and can have atleast 90%, preferably at least 95%, or more, sequence homology to theircorresponding sequences in the genome.

The exogenous sequence of interest of the invention may be introducedinto the cell by any of the means previously discussed. In a particularembodiment, the exogenous sequence of interest is introduced by way of aviral vector, such as a lentivirus, retrovirus, adenovirus, orpreferably a recombinant AAV vector. Recombinant AAV vectors useful forintroducing an exogenous nucleic acid can have any serotype that allowsfor transduction of the virus into the cell and insertion of theexogenous nucleic acid sequence into the cell genome. In particularembodiments, the recombinant AAV vectors have a serotype of AAV2 orAAV6. The recombinant AAV vectors can also be self-complementary suchthat they do not require second-strand DNA synthesis in the host cell.

In another particular embodiment, the exogenous sequence of interest canbe introduced into the cell using a single-stranded DNA template. Thesingle-stranded DNA can comprise the exogenous sequence of interest and,in preferred embodiments, can comprise 5′ and 3′ homology arms topromote insertion of the nucleic acid sequence into the nucleasecleavage site by homologous recombination. The single-stranded DNA canfurther comprise a 5′ AAV inverted terminal repeat (ITR) sequence 5′upstream of the 5′ homology arm, and a 3′ AAV ITR sequence 3′ downstreamof the 3′ homology arm.

In another particular embodiment, genes encoding an engineered nucleaseof the invention and/or an exogenous sequence of interest of theinvention can be introduced into the cell by transfection with alinearized DNA template. In some examples, a plasmid DNA can be digestedby one or more restriction enzymes such that the circular plasmid DNA islinearized prior to transfection into the cell.

T cells modified by the present invention may require activation priorto introduction of a nuclease and/or an exogenous sequence of interest.For example, T cells can be contacted with anti-CD3 and anti-CD28antibodies that are soluble or conjugated to a support (i.e., beads) fora period of time sufficient to activate the cells.

Genetically-modified cells of the invention can be further modified toexpress one or more inducible suicide genes, the induction of whichprovokes cell death and allows for selective destruction of the cells invitro or in vivo. In some examples, a suicide gene can encode acytotoxic polypeptide, a polypeptide that has the ability to convert anon-toxic pro-drug into a cytotoxic drug, and/or a polypeptide thatactivates a cytotoxic gene pathway within the cell. That is, a suicidegene is a nucleic acid that encodes a product that causes cell death byitself or in the presence of other compounds. A representative exampleof such a suicide gene is one that encodes thymidine kinase of herpessimplex virus. Additional examples are genes that encode thymidinekinase of varicella zoster virus and the bacterial gene cytosinedeaminase that can convert 5-fluorocytosine to the highly toxic compound5-fluorouracil. Suicide genes also include as non-limiting examplesgenes that encode Cas-9, Cas-8, or cytosine deaminase. In some examples,Cas-9 can be activated using a specific chemical inducer of dimerization(CID). A suicide gene can also encode a polypeptide that is expressed atthe surface of the cell that makes the cells sensitive to therapeuticand/or cytotoxic monoclonal antibodies. In further examples, a suicidegene can encode recombinant antigenic polypeptide comprising anantigenic motif recognized by the anti-CD20 mAb Rituximab and an epitopethat allows for selection of cells expressing the suicide gene. See, forexample, the RQR8 polypeptide described in WO2013153391, which comprisestwo Rituximab-binding epitopes and a QBEnd10-binding epitope. For such agene, Rituximab can be administered to a subject to induce celldepletion when needed. In further examples, a suicide gene may include aQBEnd10-binding epitope expressed in combination with a truncated EGFRpolypeptide.

T cells modified by the methods and compositions described herein canhave reduced expression of an endogenous T cell receptor and,optionally, can further express a protein of interest (e.g., a CAR).Thus, the invention further provides a population of T cells thatexpress the protein of interest and do not express the endogenous T cellreceptor. For example, the population can include a plurality ofgenetically-modified T cells of the invention which express a CAR (i.e.,are CAR+), or an exogenous T cell receptor (i.e., exoTCR+), and havereduced expression of an endogenous T cell receptor (i.e., are TCR−). Invarious embodiments of the invention, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or upto 100%, of cells in the population are a genetically-modified T cell asdescribed herein. In a particular example, the population can compriseat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or up to 100%, cells that are both TCR− andCAR+.

2.4 Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a genetically-modified T cell of the invention, or apopulation of genetically-modified T cells of the invention, and apharmaceutically-acceptable carrier. Such pharmaceutical compositionscan be prepared in accordance with known techniques. See, e.g.,Remington, The Science And Practice of Pharmacy (21^(st) ed. 2005). Inthe manufacture of a pharmaceutical formulation according to theinvention, cells are typically admixed with a pharmaceuticallyacceptable carrier and the resulting composition is administered to asubject. The carrier must, of course, be acceptable in the sense ofbeing compatible with any other ingredients in the formulation and mustnot be deleterious to the subject. In some embodiments, pharmaceuticalcompositions of the invention can further comprise one or moreadditional agents useful in the treatment of a disease in the subject.In additional embodiments, pharmaceutical compositions of the inventioncan further include biological molecules, such as cytokines (e.g., IL-2,IL-7, IL-15, and/or IL-21), which promote in vivo cell proliferation andengraftment of genetically-modified T cells. Pharmaceutical compositionscomprising genetically-modified T cells of the invention can beadministered in the same composition as an additional agent orbiological molecule or, alternatively, can be co-administered inseparate compositions.

The present disclosure also provides genetically-modified cells, orpopulations thereof, described herein for use as a medicament. Thepresent disclosure further provides the use of genetically-modifiedcells or populations thereof described herein in the manufacture of amedicament for treating a disease in a subject in need thereof. In onesuch aspect, the medicament is useful for cancer immunotherapy insubjects in need thereof.

Pharmaceutical compositions of the invention can be useful for treatingany disease state that can be targeted by T cell adoptive immunotherapy.Non-limiting examples of cancer which may be treated with thepharmaceutical compositions and medicaments of the present disclosureare carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias,and germ cell tumors, including but not limited to cancers of B-cellorigin, neuroblastoma, osteosarcoma, prostate cancer, renal cellcarcinoma, rhabdomyosarcoma, liver cancer, gastric cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, breastcancer, lung cancer, cutaneous or intraocular malignant melanoma, renalcancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system, cancer ofthe thyroid gland, cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer ofthe penis, solid tumors of childhood, lymphocytic lymphoma, cancer ofthe bladder, cancer of the kidney or ureter, carcinoma of the renalpelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cellcancer, environmentally induced cancers including those induced byasbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin's lymphomas,acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoidleukemia, immunoblastic large cell lymphoma, acute lymphoblasticleukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-celllymphoma, and any combinations of said cancers. In certain embodiments,cancers of B-cell origin include, without limitation, B-lineage acutelymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-celllymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatricindication), mantle cell lymphoma, follicular lymphoma, marginal zonelymphoma, Burkitt's lymphoma, multiple myeloma, and B-cell non-Hodgkin'slymphoma.

In some of these embodiments wherein cancer is treated with thepresently disclosed genetically-modified cells, the subject administeredthe genetically-modified cells is further administered an additionaltherapeutic, such as radiation, surgery, or a chemotherapeutic agent.

The invention further provides a population of genetically-modifiedcells comprising a plurality of genetically-modified cells describedherein, which comprise in their genome an exogenous nucleic acidmolecule encoding a sequence of interest, wherein the exogenous nucleicacid molecule is inserted into the targeted 5′ intron of the T cellreceptor alpha gene, and wherein cell-surface expression of theendogenous TCR is reduced. Thus, in various embodiments of theinvention, a population of genetically-modified cells is providedwherein at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or up to 100%, of cells in thepopulation are a genetically-modified cell described herein. In furtherembodiments of the invention, a population of genetically-modified cellsis provided wherein at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or up to 100%, of cellsin the population are a genetically-modified cell described herein whichfurther express a chimeric antigen receptor.

2.5 Methods of Administering Genetically-Modified Cells

Another aspect disclosed herein is the administration of thegenetically-modified T cells of the present disclosure to a subject inneed thereof. In particular embodiments, the pharmaceutical compositionsdescribed herein are administered to a subject in need thereof. Forexample, an effective amount of a population of cells can beadministered to a subject having a disease. In particular embodiments,the disease can be cancer, and administration of thegenetically-modified T cells of the invention represent animmunotherapy. The administered cells are able to reduce theproliferation, reduce the number, or kill target cells in the recipient.Unlike antibody therapies, genetically-modified T cells of the presentdisclosure are able to replicate and expand in vivo, resulting inlong-term persistence that can lead to sustained control of a disease.

Examples of possible routes of administration include parenteral, (e.g.,intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), orinfusion) administration. Moreover, the administration may be bycontinuous infusion or by single or multiple boluses. In specificembodiments, one or both of the agents is infused over a period of lessthan about 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour. Instill other embodiments, the infusion occurs slowly at first and then isincreased over time.

In some embodiments, a genetically-modified T cell of the presentdisclosure targets a tumor antigen for the purposes of treating cancer.Such cancers can include, without limitation, carcinoma, lymphoma,sarcoma, blastomas, leukemia, cancers of B cell origin, breast cancer,gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma,prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer,rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma. In specificembodiments, cancers and disorders include but are not limited to pre-BALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuselarge B cell lymphoma, salvage post allogenic bone marrowtransplantation, and the like. These cancers can be treated using acombination of CARs that target, for example, CD19, CD20, CD22, and/orROR1. In some non-limiting examples, a genetically-modified eukaryoticcell or population thereof of the present disclosure targets carcinomas,lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ celltumors, including but not limited to cancers of B-cell origin,neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma,rhabdomyosarcoma, liver cancer, gastric cancer, bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, breast cancer, lungcancer, cutaneous or intraocular malignant melanoma, renal cancer,uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer ofthe small intestine, cancer of the endocrine system, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the adrenalgland, sarcoma of soft tissue, cancer of the urethra, cancer of thepenis, solid tumors of childhood, lymphocytic lymphoma, cancer of thebladder, cancer of the kidney or ureter, carcinoma of the renal pelvis,neoplasm of the central nervous system (CNS), primary CNS lymphoma,tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitaryadenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,environmentally induced cancers including those induced by asbestos,multiple myeloma, Hodgkin lymphoma, non-Hodgkin's lymphomas, acutemyeloid lymphoma, chronic myelogenous leukemia, chronic lymphoidleukemia, immunoblastic large cell lymphoma, acute lymphoblasticleukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-celllymphoma, and any combinations of said cancers. In certain embodiments,cancers of B-cell origin include, without limitation, B-lineage acutelymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-celllymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatricindication), mantle cell lymphoma, follicular lymphoma, marginal zonelymphoma, Burkitt's lymphoma, multiple myeloma, and B-cell non-Hodgkin'slymphoma.

When an “effective amount” or “therapeutic amount” is indicated, theprecise amount to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size (ifpresent), extent of infection or metastasis, and condition of thepatient (subject). In some embodiments, a pharmaceutical compositioncomprising the genetically-modified cells described herein isadministered at a dosage of 10⁴ to 10⁹ cells/kg body weight, includingall integer values within those ranges. In further embodiments, thedosage is 10⁵ to 10⁶ cells/kg body weight, including all integer valueswithin those ranges. In some embodiments, cell compositions areadministered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In some embodiments, administration of genetically-modified T cells ofthe present disclosure reduce at least one symptom of a target diseaseor condition. For example, administration of genetically-modified Tcells of the present disclosure can reduce at least one symptom of acancer. Symptoms of cancers are well known in the art and can bedetermined by known techniques.

2.6 Methods for Producing Recombinant Viral Vectors

In some embodiments, the invention provides recombinant AAV vectors foruse in the methods of the invention. Recombinant AAV vectors aretypically produced in mammalian cell lines such as HEK-293. Because theviral cap and rep genes are removed from the vector to prevent itsself-replication to make room for the therapeutic gene(s) to bedelivered (e.g. the endonuclease gene), it is necessary to provide thesein trans in the packaging cell line. In addition, it is necessary toprovide the “helper” (e.g. adenoviral) components necessary to supportreplication (Cots D, Bosch A, Chillon M (2013) Curr. Gene Ther. 13(5):370-81). Frequently, recombinant AAV vectors are produced using atriple-transfection in which a cell line is transfected with a firstplasmid encoding the “helper” components, a second plasmid comprisingthe cap and rep genes, and a third plasmid comprising the viral ITRscontaining the intervening DNA sequence to be packaged into the virus.Viral particles comprising a genome (ITRs and intervening gene(s) ofinterest) encased in a capsid are then isolated from cells byfreeze-thaw cycles, sonication, detergent, or other means known in theart. Particles are then purified using cesium-chloride density gradientcentrifugation or affinity chromatography and subsequently delivered tothe gene(s) of interest to cells, tissues, or an organism such as ahuman patient.

Because recombinant AAV particles are typically produced (manufactured)in cells, precautions must be taken in practicing the current inventionto ensure that the site-specific endonuclease is not expressed in thepackaging cells. Because the viral genomes of the invention comprise arecognition sequence for the endonuclease, any endonuclease expressed inthe packaging cell line will be capable of cleaving the viral genomebefore it can be packaged into viral particles. This will result inreduced packaging efficiency and/or the packaging of fragmented genomes.Several approaches can be used to prevent endonuclease expression in thepackaging cells, including:

-   -   1. The endonuclease can be placed under the control of a        tissue-specific promoter that is not active in the packaging        cells. For example, if a viral vector is developed for delivery        of (an) endonuclease gene(s) to muscle tissue, a muscle-specific        promoter can be used. Examples of muscle-specific promoters        include C5-12 (Liu, et al. (2004) Hum Gene Ther. 15:783-92), the        muscle-specific creatine kinase (MCK) promoter (Yuasa, et        al. (2002) Gene Ther. 9:1576-88), or the smooth muscle 22 (SM22)        promoter (Haase, et al. (2013) BMC Biotechnol. 13:49-54).        Examples of CNS (neuron)-specific promoters include the NSE,        Synapsin, and MeCP2 promoters (Lentz, et al. (2012) Neurobiol        Dis. 48:179-88). Examples of liver-specific promoters include        albumin promoters (such as Palb), human α1-antitrypsin (such as        Pa1AT), and hemopexin (such as Phpx) (Kramer, M G et al., (2003)        Mol. Therapy 7:375-85). Examples of eye-specific promoters        include opsin, and corneal epithelium-specific K12 promoters        (Martin K R G, Klein R L, and Quigley H A (2002) Methods (28):        267-75) (Tong Y, et al., (2007) J Gene Med, 9:956-66). These        promoters, or other tissue-specific promoters known in the art,        are not highly-active in HEK-293 cells and, thus, will not        expected to yield significant levels of endonuclease gene        expression in packaging cells when incorporated into viral        vectors of the present invention. Similarly, the viral vectors        of the present invention contemplate the use of other cell lines        with the use of incompatible tissue specific promoters (i.e.,        the well-known HeLa cell line (human epithelial cell) and using        the liver-specific hemopexin promoter). Other examples of tissue        specific promoters include: synovial sarcomas PDZD4        (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart),        SLC5A12 (kidney), cholesterol regulation APOM (liver), ADPRHL1        (heart), and monogenic malformation syndromes TP73L (muscle).        (Jacox E, et al., (2010) PLoS One v.5(8):e12274).    -   2. Alternatively, the vector can be packaged in cells from a        different species in which the endonuclease is not likely to be        expressed. For example, viral particles can be produced in        microbial, insect, or plant cells using mammalian promoters,        such as the well-known cytomegalovirus- or SV40 virus-early        promoters, which are not active in the non-mammalian packaging        cells. In a preferred embodiment, viral particles are produced        in insect cells using the baculovirus system as described by        Gao, et al. (Gao, H., et al. (2007) J. Biotechnol.        131(2):138-43). An endonuclease under the control of a mammalian        promoter is unlikely to be expressed in these cells (Airenne, K        J, et al. (2013) Mol. Ther. 21(4):739-49). Moreover, insect        cells utilize different mRNA splicing motifs than mammalian        cells. Thus, it is possible to incorporate a mammalian intron,        such as the human growth hormone (HGH) intron or the SV40 large        T antigen intron, into the coding sequence of an endonuclease.        Because these introns are not spliced efficiently from pre-mRNA        transcripts in insect cells, insect cells will not express a        functional endonuclease and will package the full-length genome.        In contrast, mammalian cells to which the resulting recombinant        AAV particles are delivered will properly splice the pre-mRNA        and will express functional endonuclease protein. Haifeng Chen        has reported the use of the HGH and SV40 large T antigen introns        to attenuate expression of the toxic proteins barnase and        diphtheria toxin fragment A in insect packaging cells, enabling        the production of recombinant AAV vectors carrying these toxin        genes (Chen, H (2012) Mol Ther Nucleic Acids. 1(11): e57).    -   3. The endonuclease gene can be operably linked to an inducible        promoter such that a small-molecule inducer is required for        endonuclease expression. Examples of inducible promoters include        the Tet-On system (Clontech; Chen H., et al., (2015) BMC        Biotechnol. 15(1):4)) and the RheoSwitch system (Intrexon; Sowa        G., et al., (2011) Spine, 36(10): E623-8). Both systems, as well        as similar systems known in the art, rely on ligand-inducible        transcription factors (variants of the Tet Repressor and        Ecdysone receptor, respectively) that activate transcription in        response to a small-molecule activator (Doxycycline or Ecdysone,        respectively). Practicing the current invention using such        ligand-inducible transcription activators includes: 1) placing        the endonuclease gene under the control of a promoter that        responds to the corresponding transcription factor, the        endonuclease gene having (a) binding site(s) for the        transcription factor; and 2) including the gene encoding the        transcription factor in the packaged viral genome The latter        step is necessary because the endonuclease will not be expressed        in the target cells or tissues following recombinant AAV        delivery if the transcription activator is not also provided to        the same cells. The transcription activator then induces        endonuclease gene expression only in cells or tissues that are        treated with the cognate small-molecule activator. This approach        is advantageous because it enables endonuclease gene expression        to be regulated in a spatio-temporal manner by selecting when        and to which tissues the small-molecule inducer is delivered.        However, the requirement to include the inducer in the viral        genome, which has significantly limited carrying capacity,        creates a drawback to this approach.    -   4. In another preferred embodiment, recombinant AAV particles        are produced in a mammalian cell line that expresses a        transcription repressor that prevents expression of the        endonuclease. Transcription repressors are known in the art and        include the Tet-Repressor, the Lac-Repressor, the Cro repressor,        and the Lambda-repressor. Many nuclear hormone receptors such as        the ecdysone receptor also act as transcription repressors in        the absence of their cognate hormone ligand. To practice the        current invention, packaging cells are transfected/transduced        with a vector encoding a transcription repressor and the        endonuclease gene in the viral genome (packaging vector) is        operably linked to a promoter that is modified to comprise        binding sites for the repressor such that the repressor silences        the promoter. The gene encoding the transcription repressor can        be placed in a variety of positions. It can be encoded on a        separate vector; it can be incorporated into the packaging        vector outside of the ITR sequences; it can be incorporated into        the cap/rep vector or the adenoviral helper vector; or, most        preferably, it can be stably integrated into the genome of the        packaging cell such that it is expressed constitutively. Methods        to modify common mammalian promoters to incorporate        transcription repressor sites are known in the art. For example,        Chang and Roninson modified the strong, constitutive CMV and RSV        promoters to comprise operators for the Lac repressor and showed        that gene expression from the modified promoters was greatly        attenuated in cells expressing the repressor (Chang B D, and        Roninson I B (1996) Gene 183:137-42). The use of a non-human        transcription repressor ensures that transcription of the        endonuclease gene will be repressed only in the packaging cells        expressing the repressor and not in target cells or tissues        transduced with the resulting recombinant AAV vector.

In some embodiments, genetic transfer is accomplished via lentiviralvectors. Lentiviruses, in contrast to other retroviruses, in somecontexts may be used for transducing certain non-dividing cells.Non-limiting examples of lentiviral vectors include those derived from alentivirus, such as Human Immunodeficiency Virus 1 (HIV-1), HIV-2, anSimian Immunodeficiency Virus (SIV), Human T-lymphotropic virus 1(HTLV-1), HTLV-2 or equine infection anemia virus (E1AV). For example,lentiviral vectors have been generated by attenuating the HIV virulencegenes, for example, the genes env, vif, vpr, vpu and nef are deleted,making the vector safer for therapeutic purposes. Lentiviral vectors areknown in the art, see Naldini et al., (1996 and 1998); Zufferey et al.,(1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136). Insome embodiments, these viral vectors are plasmid-based or virus-based,and are configured to carry the essential sequences for incorporatingforeign nucleic acid, for selection, and for transfer of the nucleicacid into a host cell. Known lentiviruses can be readily obtained fromdepositories or collections such as the American Type Culture Collection(“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209), or isolatedfrom known sources using commonly available techniques.

In specific embodiments, lentiviral vectors are prepared using a plasmidencoding the gag, pol, tat, and rev genes cloned from humanimmunodeficiency virus (HIV) and a second plasmid encoding the envelopeprotein from vesicular stomatitis virus (VSV-G) used to pseudotype viralparticles. A transfer vector, such as the pCDH-EF1-MCS vector, can beused with a suitable promoter, if needed, and a coding sequence. Allthree plasmids can then be transfected into lentivirus cells, such asthe Lenti-X-293T cells, and lentivirus can then be harvested,concentrated and screened after a suitable incubation time. Accordingly,methods are provided herein for producing recombinant lentiviral vectorscomprising the exogenous sequence of interest described herein or anengineered nuclease of the invention.

2.7 Engineered Nuclease Variants

Embodiments of the invention encompass the engineered nucleases, andparticularly the engineered meganucleases, described herein, andvariants thereof. Further embodiments of the invention encompassisolated polynucleotides comprising a nucleic acid sequence encoding theengineered meganucleases described herein, and variants of suchpolynucleotides.

As used herein, “variants” is intended to mean substantially similarsequences. A “variant” polypeptide is intended to mean a polypeptidederived from the “native” polypeptide by deletion or addition of one ormore amino acids at one or more internal sites in the native proteinand/or substitution of one or more amino acids at one or more sites inthe native polypeptide. As used herein, a “native” polynucleotide orpolypeptide comprises a parental sequence from which variants arederived. Variant polypeptides encompassed by the embodiments arebiologically active. That is, they continue to possess the desiredbiological activity of the native protein; i.e., the ability torecognize and cleave recognition sequences found in the targeted 5′intron of the human T cell receptor alpha gene, including, for example,the TRC 11-12 recognition sequence (SEQ ID NO: 4), the TRC 15-16recognition sequence (SEQ ID NO: 6), the TRC 17-18 recognition sequence(SEQ ID NO: 8), and the TRC 19-20 recognition sequence (SEQ ID NO: 10).Such variants may result, for example, from human manipulation.Biologically active variants of a native polypeptide of the embodiments(e.g., SEQ ID NOs: 12-27), or biologically active variants of therecognition half-site binding subunits described herein (e.g., SEQ IDNOs: 28-59), will have at least about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, or about 99%, sequence identity to the aminoacid sequence of the native polypeptide or native subunit, as determinedby sequence alignment programs and parameters described elsewhereherein. A biologically active variant of a polypeptide or subunit of theembodiments may differ from that polypeptide or subunit by as few asabout 1-40 amino acid residues, as few as about 1-20, as few as about1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acidresidue.

The polypeptides of the embodiments may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants can be prepared bymutations in the DNA. Methods for mutagenesis and polynucleotidealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be optimal.

A substantial number of amino acid modifications to the DNA recognitiondomain of the wild-type I-CreI meganuclease have previously beenidentified (e.g., U.S. Pat. No. 8,021,867) which, singly or incombination, result in engineered meganucleases with specificitiesaltered at individual bases within the DNA recognition sequencehalf-site, such that the resulting rationally-designed meganucleaseshave half-site specificities different from the wild-type enzyme. Table5 provides potential substitutions that can be made in a recombinantmeganuclease monomer or subunit to enhance specificity based on the basepresent at each half-site position (−1 through −9) of a recognitionhalf-site.

TABLE 5 Favored Sense-Strand Base Posn. A C G T A/T A/C A/G C/T G/TA/G/T A/C/G/T −1 Y75  R70* K70  Q70*  T46* G70  L75*  H75*  E70* C70 A70 C75*  R75*  E75* L70 S70 Y139*  H46*  E46*  Y75*  G46*  C46*  K46* D46*  Q75*  A46*  R46*  H75*  H139  Q46*  H46* −2 Q70 E70 H70  Q44* C44*  T44* D70  D44*  A44*  K44*  E44*  V44*  R44*   I44*  L44*  N44*−3 Q68 E68 R68 M68 H68 Y68 K68  C24* F68 C68   I24*  K24* L68  R24* F68−4  A26* E77 R77 S77  S26* Q77  K26*  E26*  Q26* −5 E42 R42  K28*  C28*M66 Q42 K66 −6 Q40 E40 R40 C40 A40 S40  C28*  R28*  I40 A79  S28* V40 A28* C79  H28*  I79 V79  Q28* −7  N30* E38 K38  I38 C38 H38 Q38  K30*R38 L38 N38  R30*  E30*  Q30* −8 F33 E33 F33 L33  R32* R33 Y33 D33 H33V33  I33 F33 C33 −9 E32 R32 L32 D32 S32 K32 V32  I32 N32 A32 H32 C32 Q32T32 Bold entries are wild-type contact residues and do not constitute“modifications” as used herein. An asterisk indicates that the residuecontacts the base on the antisense strand.

For polynucleotides, a “variant” comprises a deletion and/or addition ofone or more nucleotides at one or more sites within the nativepolynucleotide. One of skill in the art will recognize that variants ofthe nucleic acids of the embodiments will be constructed such that theopen reading frame is maintained. For polynucleotides, conservativevariants include those sequences that, because of the degeneracy of thegenetic code, encode the amino acid sequence of one of the polypeptidesof the embodiments. Variant polynucleotides include syntheticallyderived polynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a recombinantmeganuclease of the embodiments. Generally, variants of a particularpolynucleotide of the embodiments will have at least about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99% or moresequence identity to that particular polynucleotide as determined bysequence alignment programs and parameters described elsewhere herein.Variants of a particular polynucleotide of the embodiments (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the polypeptide. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by screening the polypeptide for its ability topreferentially recognize and cleave recognition sequences found withinthe targeted 5′ intron of the human T cell receptor alpha gene.

EXAMPLES

This invention is further illustrated by the following examples, whichshould not be construed as limiting. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.

Example 1 Characterization of Meganucleases that Recognize and CleaveRecognition Sequences in the Targeted 5′ Intron of the T Cell ReceptorAlpha Gene

1. Meganucleases that Recognize and Cleave the TRC 11-12 RecognitionSequence

Engineered meganucleases (SEQ ID NOs: 12-15), collectively referred toherein as “TRC 11-12 meganucleases,” were engineered to recognize andcleave the TRC 11-12 recognition sequence (SEQ ID NO: 4), which ispresent in the targeted 5′ intron of the human T cell receptor alphagene. Each TRC 11-12 recombinant meganuclease comprises an N-terminalnuclear-localization signal derived from SV40, a first meganucleasesubunit, a linker sequence, and a second meganuclease subunit. A firstsubunit in each TRC 11-12 meganuclease binds to the TRC12 recognitionhalf-site of SEQ ID NO: 4, while a second subunit binds to the TRC11recognition half-site (see, FIG. 2).

The TRC12-binding subunits and TRC11-binding subunits each comprise a 56base pair hypervariable region, referred to as HVR1 and HVR2,respectively. TRC12-binding subunits are highly conserved outside of theHVR1 region. Similarly, TRC11-binding subunits are also highly conservedoutside of the HVR2 region. The TRC11-binding regions of SEQ ID NOs:12-15 are provided as SEQ ID NOs: 28-31, respectively. Each of SEQ IDNOs: 28-31 share at least 90% sequence identity to SEQ ID NO: 28, whichis the TRC11-binding region of the meganuclease TRC 11-12x.4 (SEQ ID NO:12). TRC12-binding regions of SEQ ID NOs: 12-15 are provided as SEQ IDNOs: 32-35, respectively. Each of SEQ ID NOs: 32-35 share at least 90%sequence identity to SEQ ID NO: 32, which is the TRC12-binding region ofthe meganuclease TRC 11-12x.4 (SEQ ID NO: 12).

2. Meganucleases that Recognize and Cleave the TRC 15-16 RecognitionSequence

Engineered meganucleases (SEQ ID NOs: 16-19), collectively referred toherein as “TRC 15-16 meganucleases,” were engineered to recognize andcleave the TRC 15-16 recognition sequence (SEQ ID NO: 6), which ispresent in the targeted 5′ intron of the human T cell receptor alphagene. Each TRC 15-16 recombinant meganuclease comprises an N-terminalnuclear-localization signal derived from SV40, a first meganucleasesubunit, a linker sequence, and a second meganuclease subunit. A firstsubunit in each TRC 15-16 meganuclease binds to the TRC15 recognitionhalf-site of SEQ ID NO: 6, while a second subunit binds to the TRC16recognition half-site (see, FIG. 2).

The TRC15-binding subunits and TRC16-binding subunits each comprise a 56base pair hypervariable region, referred to as HVR1 and HVR2,respectively. TRC15-binding subunits are highly conserved outside of theHVR1 region. Similarly, TRC16-binding subunits are also highly conservedoutside of the HVR2 region. The TRC15-binding regions of SEQ ID NOs:16-19 are provided as SEQ ID NOs: 36-39, respectively. Each of SEQ IDNOs: 36-39 share at least 90% sequence identity to SEQ ID NO: 36, whichis the TRC15-binding region of the meganuclease TRC 15-16x.31 (SEQ IDNO: 16). TRC16-binding regions of SEQ ID NOs: 16-19 are provided as SEQID NOs: 40-43, respectively. Each of SEQ ID NOs: 40-43 share at least90% sequence identity to SEQ ID NO: 40, which is the TRC16-bindingregion of the meganuclease TRC 15-16x.31 (SEQ ID NO: 16).

3. Meganucleases that Recognize and Cleave the TRC 17-18 RecognitionSequence

Engineered meganucleases (SEQ ID NOs: 20-23), collectively referred toherein as “TRC 17-18 meganucleases,” were engineered to recognize andcleave the TRC 17-18 recognition sequence (SEQ ID NO: 8), which ispresent in the targeted 5′ intron of the human T cell receptor alphagene. Each TRC 17-18 recombinant meganuclease comprises an N-terminalnuclear-localization signal derived from SV40, a first meganucleasesubunit, a linker sequence, and a second meganuclease subunit. A firstsubunit in each TRC 17-18 meganuclease binds to the TRC17 recognitionhalf-site of SEQ ID NO: 8, while a second subunit binds to the TRC18recognition half-site (see, FIG. 2).

The TRC17-binding subunits and TRC18-binding subunits each comprise a 56base pair hypervariable region, referred to as HVR1 and HVR2,respectively. TRC17-binding subunits are highly conserved outside of theHVR1 region. Similarly, TRC18-binding subunits are also highly conservedoutside of the HVR2 region. The TRC17-binding regions of SEQ ID NOs:20-23 are provided as SEQ ID NOs: 44-47, respectively. Each of SEQ IDNOs: 44-47 share at least 90% sequence identity to SEQ ID NO: 44, whichis the TRC17-binding region of the meganuclease TRC17-18x.15 (SEQ ID NO:20). TRC18-binding regions of SEQ ID NOs: 20-23 are provided as SEQ IDNOs: 48-51, respectively. Each of SEQ ID NOs: 48-51 share at least 90%sequence identity to SEQ ID NO: 48, which is the TRC18-binding region ofthe meganuclease TRC17-18x.15 (SEQ ID NO: 20).

4. Meganucleases that Recognize and Cleave the TRC 19-20 RecognitionSequence

Engineered meganucleases (SEQ ID NOs: 24-27), collectively referred toherein as “TRC 19-20 meganucleases,” were engineered to recognize andcleave the TRC 19-20 recognition sequence (SEQ ID NO: 10), which ispresent in the targeted 5′ intron of the human T cell receptor alphagene. Each TRC 19-20 recombinant meganuclease comprises an N-terminalnuclear-localization signal derived from SV40, a first meganucleasesubunit, a linker sequence, and a second meganuclease subunit. A firstsubunit in each TRC 19-20 meganuclease binds to the TRC19 recognitionhalf-site of SEQ ID NO: 10, while a second subunit binds to the TRC20recognition half-site (see, FIG. 2).

The TRC19-binding subunits and TRC20-binding subunits each comprise a 56base pair hypervariable region, referred to as HVR1 and HVR2,respectively. TRC19-binding subunits are highly conserved outside of theHVR1 region. Similarly, TRC20-binding subunits are also highly conservedoutside of the HVR2 region. The TRC19-binding regions of SEQ ID NOs:24-27 are provided as SEQ ID NOs: 52-55, respectively. Each of SEQ IDNOs: 52-55 share at least 90% sequence identity to SEQ ID NO: 52, whichis the TRC19-binding region of the meganuclease TRC 19-20x.85 (SEQ IDNO: 24). TRC20-binding regions of SEQ ID NOs: 24-27 are provided as SEQID NOs: 56-59, respectively. Each of SEQ ID NOs: 56-59 share at least90% sequence identity to SEQ ID NO: 56, which is the TRC20-bindingregion of the meganuclease TRC 19-20x.85 (SEQ ID NO: 24).

5. Cleavage of TRC Recognition Sequences in a CHO Cell Reporter Assay

To determine whether TRC 11-12, TRC 15-16, TRC 17-18, and TRC 19-20meganucleases could recognize and cleave their respective recognitionsequences (SEQ ID NOs: 4, 6, 8, and 10, respectively), each recombinantmeganuclease was evaluated using the CHO cell reporter assay previouslydescribed (see, WO/2012/167192 and FIG. 4). To perform the assays, CHOcell reporter lines were produced which carried a non-functional GreenFluorescent Protein (GFP) gene expression cassette integrated into thegenome of the cells. The GFP gene in each cell line was interrupted by apair of recognition sequences such that intracellular cleavage of eitherrecognition sequence by a meganuclease would stimulate a homologousrecombination event resulting in a functional GFP gene.

In CHO reporter cell lines developed for this study, one recognitionsequence inserted into the GFP gene was the TRC 11-12 recognitionsequence (SEQ ID NO: 4), the TRC 15-16 recognition sequence (SEQ ID NO:6), the TRC 17-18 recognition sequence (SEQ ID NO: 8), or the TRC 19-20recognition sequence (SEQ ID NO: 10). The second recognition sequenceinserted into the GFP gene was a CHO-23/24 recognition sequence, whichis recognized and cleaved by a control meganuclease called “CHO-23/24”.CHO reporter cells comprising the TRC 11-21 recognition sequence and theCHO-23/24 recognition sequence are referred to herein as “TRC 11-12cells.” CHO reporter cells comprising the TRC 15-16 recognition sequenceand the CHO-23/24 recognition sequence are referred to herein as “TRC15-16 cells.” CHO reporter cells comprising the TRC 17-18 recognitionsequence and the CHO-23/24 recognition sequence are referred to hereinas “TRC 17-18 cells.” CHO reporter cells comprising the TRC 19-20recognition sequence and the CHO-23/24 recognition sequence are referredto herein as “TRC 19-20 cells.”

CHO reporter cells were transfected with plasmid DNA encoding theircorresponding engineered meganucleases (e.g., TRC 11-12 cells weretransfected with plasmid DNA encoding TRC 11-12 meganucleases) orencoding the CHO-23/34 meganuclease. In each assay, 4e⁵ CHO reportercells were transfected with 50 ng of plasmid DNA in a 96-well plateusing Lipofectamine 2000 (ThermoFisher) according to the manufacturer'sinstructions. At 48 hours post-transfection, cells were evaluated byflow cytometry to determine the percentage of GFP-positive cellscompared to an untransfected negative control (bs). As shown in FIGS.5A, 5B, 5C, and 5D, all TRC meganucleases were found to produceGFP-positive cells in cell lines comprising their correspondingrecognition sequence at frequencies significantly exceeding the negativecontrol.

The efficacy of the TRC 11-12, TRC 15-16, TRC 17-18, and TRC 19-20engineered meganucleases was also determined in a time-dependent manner2, 5, and 7 days after introduction of the meganucleases into theircorresponding reporter cell line. In this study, each reporter cell line(1.0×10⁶ cells) was electroporated with 1×10⁶ copies of thecorresponding meganuclease mRNA per cell using a BioRad Gene PulserXcell according to the manufacturer's instructions. At the specifiedtime intervals, cells were evaluated by flow cytometry to determine thepercentage of GFP-positive cells. As shown in FIGS. 6A, 6B, 6C, and 6D,% GFP expression varied among the TRC 11-12, TRC 15-16, TRC 17-18, andTRC 19-20 meganucleases, with some maintaining approximately the same %GFP throughout the course of the study, while others showed a decreasein % GFP expression after 5 or 7 days.

6. Conclusions

These studies demonstrated that TRC 11-12 meganucleases, TRC 15-16meganucleases, TRC 17-18 meganucleases, and TRC 19-20 meganucleasesencompassed by the invention can efficiently target and cleave theirrespective recognition sequences in cells.

Example 2 Generation of Indels at TRC Recognition Sequences in Human TCells 1. Background

This study demonstrated that engineered nucleases encompassed by theinvention could cleave their respective recognition sequences in human Tcells. Human CD3+ T cells were isolated from PBMCs by magneticseparation and activated for 72 hours using antibodies against CD3 andCD28. 1e⁶ activated human T cells were electroporated with 2e⁶ copies ofa given TRC 11-12 or TRC 15-16 meganuclease mRNA per cell using a Lonza4D-Nucleofector according to the manufacturer's instructions. At 72hours post-transfection, genomic DNA (gDNA) was harvested from cells anda T7 endonuclease I (T7E) assay was performed to estimate geneticmodification at the endogenous TRC 11-12 or TRC 15-16 recognitionsequence (FIG. 7). In the T7E assay, the TRC 11-12 or TRC 15-6 locus isamplified by PCR using primers that flank the two recognition sequences.If there are indels (random insertions or deletions) within the targetlocus, the resulting PCR product will consist of a mix of wild-typealleles and mutant alleles. The PCR product is denatured and allowed toslowly reanneal. Slow reannealing allows for the formation ofheteroduplexes consisting of wild-type and mutant alleles, resulting inmismatched bases and/or bulges. The T7E1 enzyme cleaves at mismatchsites, resulting in cleavage products that can be visualized by gelelectrophoresis.

2. Results

Mock-electroporated cells and control gDNA (Lanes 1 and 2, respectively)both show a single band corresponding to the full-length PCR with noT7E-digested bands, indicating no indels or other polymorphisms arepresent in either the TRC 11-12 or TRC 15-16 recognition sequences (FIG.7). Lanes 3, 5 and 6, corresponding to cells electroporated with TRC11-12x.4, TRC 11-12x.63, and TRC 11-12x.82, respectively, show thefull-length PCR band along with smaller T7E-digested bands indicative ofindels within the recognition site. Lane 4, corresponding to cellselectroporated with TRC 11-12x.60, only showed a full-length PCR band,indicating no indels at the recognition site. Lanes 7, 8, and 10,corresponding to cells electroporated with TRC 15-16x.31, TRC 15-16x.87,and TRC 15-16x.89, respectively, show the full-length PCR band alongwith smaller T7E-digested bands indicative of indels within therecognition site. Lane 9, corresponding to cells electroporated with TRC15-16x.63, only showed a full-length PCR band, indicating no indels atthe recognition site.

3. Conclusions

These data demonstrate that several TRC 11-12 and TRC 15-16 nucleasesare able to cleave their respective recognition sequences in human Tcells. Cells electroporated with either TRC 11-12x.4, TRC 11-12x.63, TRC11-12x.82, TRC 15-16x.31, TRC 15-16x.87, or TRC 15-16x.89 all showedT7E-digested bands, demonstrating the presences of indels in theirrespective recognition sequences.

Example 3 Effect of TRC Recognition Sequence Cleavage on T Cell ReceptorExpression 1. Background

The purpose of these experiments was to demonstrate whether cleavage ofa TRC recognition sequence within the targeted 5′ intron of the T cellreceptor alpha gene, and subsequent repair by NHEJ, would affectexpression of the endogenous T cell receptor.

Human T cells were magnetically enriched using a CD3 positive selectionkit and a Robo-Sep automated magnetic separator (both from Stem CellTechnologies). T cells were enriched from an apheresis sample obtainedfrom a compensated, healthy human volunteer. T cells were stimulated for3 days using T cell Activator (antiCD3/anti/CD28) Dynabeads(LifeTechnologies) at a 1:1 cell:bead ratio in the presence of 10 ng/mlif IL-2. After 3 days, T cells were harvested, Dynabeads were removedusing the DynaMag magnet (Life Technologies), and 1 μg of the indicatedmeganuclease RNA was introduced to T cell samples using the Lonza 4-Dnucleofector. Nucleofected cells were cultured for 6 days prior to flowcytometric analysis. CD3 surface display was measured by labeling T cellsamples with 10 of anti-CD3-BrilliantViolet711 (BioLegend product300464) and 0.3 μl of GhostDye-510 (Tonbo Biosciences) per sample of2.0×10⁵ cells. Data were acquired using a Beckman-Coulter CytoFLEX-Scytometer.

2. Results

T cells were nucleofected with either TRC 1-2x.87EE (an engineerednuclease which targets TRAC exon 1) or no RNA (mock) to serve aspositive and negative controls for TRAC locus editing, respectively, andappear in FIG. 8A and FIG. 8B. Four additional samples were alsonucleofected with RNA encoding one distinct nuclease variant from theTRC 15-16 family, all members of which target the TRC 15-16 recognitionsequence in the 5′ intron. When TRAC locus editing results in genedisruption, no TCRα chains are synthesized, and no TCR complex(including CD3) is displayed on the surface of edited cells. Greaterthan half of the TRC 1-2x.87EE edited T cells were shown to be TRCnegative due to cleavage in exon 1 and error-prone repair of thecleavage site by NHEJ (FIG. 8B). By comparison, the frequency of TCRnegative cells following editing by TRC 15-16x.31, TRC 15-16x.63, TRC15-16x.87, and TRC 15-16x.89 was between only 4% and 8% (FIGS. 8C, 8D,8E, and 8F, respectively).

3. Conclusions

These experiments demonstrate that targeting recognition sequences inthe 5′ intron upstream of TRAC exon 1 can produce indels (as observed inthe T7E assay) but do not substantially affect cell surface expressionof the endogenous T cell receptor. We expect that insertion of aconstruct into this cleavage site, which comprises an exogenous spliceacceptor site, a CAR coding sequence, and a polyA signal, will knock outTCR expression in cells.

Example 4 Insertion of a Sequence of Interest into the Targeted 5′Intron

The purpose of these experiments is to generate a double strand cleavagein the targeted 5′ intron and to insert an exogenous sequence ofinterest into the cleavage site by homologous recombination, thusallowing for: (i) disrupted expression of the endogenous T cell receptordue to the presence of an exogenous splice acceptor site and/or a poly Asignal, and (ii) expression of a protein of interest encoded by theinsert.

In these examples, the exogenous sequence of interest includes a numberof elements which are shown in the constructs of FIG. 9. Each constructis flanked by a 5′ homology arm and a 3′ homology arm. These arms havehomology to the sequences 5′ upstream and 3′ downstream of theirrespective TRC recognition sequences in the targeted 5′ intron. The sizeof each homology and, and the percent homology of the arm to thecorresponding sequence in the targeted 5′ intron, can be modulated asneeded to improve homologous recombination of the construct into thecleavage site. Adjacent to the 5′ homology arm is a chimeric intronwhich comprises both an exogenous branch site for splicing and anexogenous splice acceptor site. A T2A element is then included 5′ to ananti-CD19 CAR sequence, which includes a signal peptide, an anti-CD19scFv, a CD8 hinge and transmembrane domain, an N6 co-stimulatory domain,and a CD3t signaling domain. The CAR coding sequence is followed by abi-polyA signal, and finally a 3′ homology arm. The specific constructsprovided in FIGS. 9A-9C target the TRC 11-12, TRC 15-16, and TRC 17-18recognition sequences, respectively, and are provided in SEQ ID NOs:60-62.

Donor human T cells can be obtained, activated, and transfected with TRCmeganuclease mRNA as described in the previous Examples. The donortemplate comprising the exogenous sequence of interest (e.g., SEQ IDNOs: 60-62, FIGS. 9A-9C) can be introduced by a number of means known inthe art, but preferably by transduction of a recombinant AAV whichcomprises the donor template. Transduction can be performed at any timerelative to transfection with the meganuclease mRNA, but preferablytransduction and transfection are performed at the same time. The levelof T cell receptor expression, and the level of CAR expression, willeach be determined in the cells over a number of time points by flowcytometry (as described above and by methods known in the art). It isexpected that the vast majority of TCR− cells obtained by this methodwill also be CAR+, as any cells which do not have the insert at thecleavage site will continue to express the endogenous TCR.

In a particular study, a promoterless GFP or CAR coding sequence wasinserted into the targeted 5′ intron to demonstrate that the endogenousTCR promoter could drive expression of these proteins when they wereintroduced using a construct of the invention. In this study, anapheresis sample was drawn from a healthy, informed, and compensateddonor, and the T cells were enriched using the CD3 positive selectionkit II in accord with the manufacturer's instructions (Stem CellTechnologies). T cells were activated using ImmunoCult T cell stimulator(anti-CD2/CD3/CD28—Stem Cell Technologies) in X-VIVO 15 medium (Lonza)supplemented with 5% fetal bovine serum and 10 ng/ml IL-2 (Gibco). After3 days of stimulation, cells were collected and RNA encoding one of twoTRC nucleases was introduced to the T cells by way of electroporationwith the 4-D Nucleofector (Lonza). T cells received RNA encoding eitherTRC 11-12x.82 or TRC 15-16x.31.

Cells receiving TRC 11-12x.82 RNA were transduced with one of two AAV6vectors containing regions of homology to genomic sequences flanking theTRC 11-12x.82 recognition sequence (i.e., the TRC 11-12 recognitionsequence). One vector, containing construct 7227 (SEQ ID NO: 63),encodes a T2A sequence followed by a promoterless GFP gene, while theother vector contains construct 7225 (SEQ ID NO: 64), which encodes aT2A sequence followed by a promoterless CAR gene.

Cells receiving TRC 15-16.x31 RNA were transduced with one of two AAV6vectors containing regions of homology to genomic sequences flanking theTRC 15-16x.31 recognition sequence (i.e., the TRC 15-16 recognitionsequence). One vector, containing construct 7228 (SEQ ID NO: 65),encodes a T2A sequence followed by a promoterless GFP gene, while theother vector contains construct 7226 (SEQ ID NO: 66), which encodes aT2A sequence followed by a promoterless CAR gene.

All transductions were carried out with a multiplicity of infection(MOI) of 50,000 (viral genomes/cell). Cell cultures were maintained for5 additional days in X-VIVO15 medium supplemented with 5% FBS and 30ng/ml of IL-2. On day 5, analyses of TRC knockout, and GFP or CARknock-in, were performed by staining cells for CD3 (Anti-CD3-APC/750 or-BV711, BioLegend) and CAR (anti-FMC63-biotin+streptavidin-PE, producedin-house) and measuring signal with a Beckman-Coulter CytoFLEX-S flowcytometer.

2. Results

The frequencies of TCR knockout cells (CD3+ vs CD3− frequencies) and GFPknock-in cells appear in FIG. 10. Following administration of TRC11-12x.82 and AAV6-7227, 18% of all T cells in culture are CD3−GFP+(FIG. 10A). When gating on only the TCR-edited (CD3−) population, 85% ofcells were GFP+ (FIG. 10B). Administration of TRC 15-16x.31 andAAV6-7228 produced a slightly lower frequency of CD3−/GFP+ cells (12.6%,FIG. 10C), although the frequency of GFP+ cells in the CD3− populationwas still above 80% (FIG. 10D).

CAR knock-in (FMC63+) into the targeted 5′ intron is shown in FIG. 11.Relative to edited cells that were not transduced with a CAR vector,(FIG. 11A), staining samples with anti-FMC63 and anti-CD3 identifiesknockout as well as knock-in populations in vector-transduced samples(histograms in FIG. 11 are gated on CD3-negative events). Insertion ofthe promoterless CAR constructs at the TRC 11-12 and TCR 15-16recognition sequences (FIGS. 11B and 11C, respectively) each yieldedCD3-/CAR+ events, indicating that the endogenous TCR promoter droveexpression of the CAR coding sequence when inserted into the targeted 5′intron.

3. Conclusions

The observation of CD3-/GFP+, or CD3-/CAR+, events after administrationof TCR intron-specific meganucleases along with T2A-transgene constructsof corresponding homology indicates that the endogenous TCRtranscriptional control elements can be used to drive expression ofproteins of interest.

What is claimed is:
 1. An isolated genetically-modified human T cellcomprising in its genome a modified human T cell receptor alpha gene,wherein said modified human T cell receptor alpha gene comprises anexogenous sequence of interest inserted into an intron within the humanT cell receptor alpha gene that is positioned 5′ upstream of the T cellreceptor alpha constant region (TRAC) exon 1, and wherein said exogenoussequence of interest comprises an exogenous splice acceptor site or anexogenous splice acceptor site and a poly A signal, and wherein anendogenous splice donor site and an endogenous splice acceptor siteflanking said intron are unmodified, and wherein saidgenetically-modified human T cell does not express an endogenous T cellreceptor on the cell surface.
 2. The isolated genetically-modified humanT cell of claim 1, wherein said intron comprises SEQ ID NO:
 3. 3. Theisolated genetically-modified human T cell of claim 1, wherein saidexogenous sequence of interest comprises, from 5′ to 3′, an exogenoussplice acceptor site, a 2A element or IRES element, a coding sequencefor a protein of interest, and a polyA signal.
 4. The isolatedgenetically-modified human T cell of claim 3, wherein said 2A element isa T2A element.
 5. The isolated genetically-modified human T cell ofclaim 1, wherein said sequence of interest comprises a coding sequencefor a chimeric antigen receptor or an exogenous T cell receptor.
 6. Theisolated genetically-modified human T cell of claim 1, wherein saidexogenous sequence of interest is inserted into said intron at anengineered meganuclease recognition site.
 7. The isolatedgenetically-modified human T cell of claim 1, wherein said exogenoussequence of interest comprises, from 5′ to 3′, an exogenous spliceacceptor site, a 2A element or IRES element, a coding sequence for aprotein of interest, and a polyA signal, and wherein said sequence ofinterest comprises a coding sequence for a chimeric antigen receptor oran exogenous T cell receptor.
 8. The isolated genetically-modified humanT cell of claim 7, wherein said exogenous sequence of interest isinserted into said intron at an engineered meganuclease recognitionsite.
 9. An isolated population of genetically-modified human T cellscomprising a plurality of said isolated genetically-modified human Tcell of claim
 1. 10. An isolated population of genetically-modifiedhuman T cells comprising a plurality of said isolatedgenetically-modified human T cell of claim
 3. 11. An isolated populationof genetically-modified human T cells comprising a plurality of saidisolated genetically-modified human T cell of claim
 5. 12. An isolatedpopulation of genetically-modified human T cells comprising a pluralityof said isolated genetically-modified human T cell of claim
 7. 13. Apharmaceutical composition useful for treatment of a disease in asubject in need thereof, wherein said pharmaceutical compositioncomprises a pharmaceutically-acceptable carrier and said isolatedgenetically-modified human T cell of claim
 1. 14. A pharmaceuticalcomposition useful for treatment of a disease in a subject in needthereof, wherein said pharmaceutical composition comprises apharmaceutically-acceptable carrier and said isolatedgenetically-modified human T cell of claim
 3. 15. A pharmaceuticalcomposition useful for treatment of a disease in a subject in needthereof, wherein said pharmaceutical composition comprises apharmaceutically-acceptable carrier and said isolatedgenetically-modified human T cell of claim
 5. 16. A pharmaceuticalcomposition useful for treatment of a disease in a subject in needthereof, wherein said pharmaceutical composition comprises apharmaceutically-acceptable carrier and said isolatedgenetically-modified human T cell of claim
 7. 17. A method of treating adisease in a subject in need thereof, said method comprisingadministering to said subject said isolated genetically-modified human Tcell of claim
 1. 18. A method of treating a disease in a subject in needthereof, said method comprising administering to said subject saidisolated genetically-modified human T cell of claim
 3. 19. A method oftreating a disease in a subject in need thereof, said method comprisingadministering to said subject said isolated genetically-modified human Tcell of claim
 5. 20. A method of treating a disease in a subject in needthereof, said method comprising administering to said subject saidisolated genetically-modified human T cell of claim 7.